NOTEBOOK

ELISA

This is a protocol for performing an ELISA assay.

Instructions on the plate reader:

1. Put tube “Liquid 1” in buffer of choice

2. Open HydroControl software

3. Place the plate on the reader

4. Choose wanted step

5. Press “start”

6. At the very end, when finished: Tube out of the liquid, select “Gerät”- “Vorfüllen”, place tube into sterile filtered ddH20, select “Gerät”- “Vorfüllen”,select “Gerät”- “Spülen”-“Nacht”, remove tube from H2O, select “Gerät”- “Vorfüllen”

Q5 PCR

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
Master mix	25 μL
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	… ng / … μL
ddH2O	… μL
Total	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	…:…, 98 °C
Denaturation	…:…, 98 °C
Annealing	…:…, … °C
Extension	…:…, 72 °C
Final extension	…:…, 72 °C
Cycles	×…

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

DH5α transformation & suspension culture of M and R

Goal

Transformation was performed to introduce plasmid DNA construct(s) … into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 200 - 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Maxiprep

Goal

A maxiprep of … was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)

ELISA

This is a protocol for performing an ELISA assay.

Instructions on the plate reader:

1. Put tube “Liquid 1” in buffer of choice

2. Open HydroControl software

3. Place the plate on the reader

4. Choose wanted step

5. Press “start”

6. At the very end, when finished: Tube out of the liquid, select “Gerät”- “Vorfüllen”, place tube into sterile filtered ddH20, select “Gerät”- “Vorfüllen”,select “Gerät”- “Spülen”-“Nacht”, remove tube from H2O, select “Gerät”- “Vorfüllen”

DH5α transformation & plating

Goal

Transformation was performed to introduce plasmid DNA construct(s) … into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Gibson assembly

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments.

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
Master mix	5 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Gibson assembly

Goal:

Seamless joining of multiple DNA fragments in a single isothermal reaction

Procedure:

Results:

DH5α transformation & suspension culture of stable construct and construct E1a, G3

Goal

Transformation was performed to introduce plasmid DNA construct(s) E, G, stable1, stable2 into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 200 - 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Sanger sequencing of R4a,b Wa,b, G3 (reseq)

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
08035649	46	R4a
50	47	R4a
51	46	R4b
52	47	R4b
53	46	W3a
54	48	W3a
55	46	W3b
56	48	W3b
08035657	64	G3

Maxiprep of construct kill switch

Goal

A maxiprep of … was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)

DH5α colony picking

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with … from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Q5 PCR of R, G

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) R,G with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	100 ng / 1 μL
ddH2O	19 μL
Total	25 μL

In the end 1 μL polymerase was added in an 1:3 dilution in ddH2O (nuclease free).

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

For R Backbone (a, b, c):

Amplification of backbone (P)

Primers C1/C2 (071/072): (6700 bp)

a, b, c:

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 70 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×34

d, e, f:

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 71 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Maxiprep

Goal

A maxiprep of … was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)

ELISA

This is a protocol for performing an ELISA assay.

Instructions on the plate reader:

1. Put tube “Liquid 1” in buffer of choice

2. Open HydroControl software

3. Place the plate on the reader

4. Choose wanted step

5. Press “start”

6. At the very end, when finished: Tube out of the liquid, select “Gerät”- “Vorfüllen”, place tube into sterile filtered ddH20, select “Gerät”- “Vorfüllen”,select “Gerät”- “Spülen”-“Nacht”, remove tube from H2O, select “Gerät”- “Vorfüllen”

Sanger sequencing of E1ab R4ab W3ab G2 G3

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

E: 46/64

R: 46/47

W: 46/47

G: 48/46/47

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
08035805	46 fw	E1a	worked
08035813	64 rv	E1a	worked
08035806	46 fw	E1b
08035814	64 rv	E1b
08035807	46fw	R4a	seems good but reseq? (better fw)
08035815	47 rv	R4a
08035808	46 fw	R4b	seems better but reseq? (better fw?)
08035816	47 rv	R4b
08035809	46 fw	W3a	fw did not work reseq with 46, 48
08035817	47 rv	W3a
08035810	46 fw	W3b	fw did not work, reseq with 46, 48
08035818	47 rv	W3b
08035811	46 fw	G2
08035819	48 fw	G2
08035821	47 rv	G2
08035812	46 fw	G3	perfect
08035820	48 fw	G3
08035822	47 rv	G3	rv too far, reseq with 64 just in case

Miniprep of W, J, R, O, R4, E1, W3, M3

Goal

A miniprep of W, J, R, O, R4, E1, W3, M3 was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)

Agarose gel electrophoresis of R repeat

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: … Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

ELISA

This is a protocol for performing an ELISA assay.

Instructions on the plate reader:

1. Put tube “Liquid 1” in buffer of choice

2. Open HydroControl software

3. Place the plate on the reader

4. Choose wanted step

5. Press “start”

6. At the very end, when finished: Tube out of the liquid, select “Gerät”- “Vorfüllen”, place tube into sterile filtered ddH20, select “Gerät”- “Vorfüllen”,select “Gerät”- “Spülen”-“Nacht”, remove tube from H2O, select “Gerät”- “Vorfüllen”

Sanger sequencing of K, K1 (maxi), D, F, G2, G3

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of K, D, F using the following primer(s):

Construct	Forward primer	Reverse primer
K	48	64,47
D	48, 46	64,47
F	46	64, 47
G2	48	64
G3	48	64
K1	48	64, 47

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
	48 fw	K
	64 rv	K
	47 rv	K
	48 fw	D
	46 fw	D
	64 rv	D
	47 rv	D
	46 fw	F
	64 rv	F
	47 rv	F
	48 fw	G2
	64 rv	G2
	48 fw	G3
	64 rv	G3
	48 fw	K1(maxi)
	64 rv	K1(maxi)
	47 rv	K1(maxi)

Q5 PCR repeat for R (Backbone)

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) R with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	100 ng / 1 μL
ddH2O	19 μL
Total	25 μL

In the end 1 μL polymerase was added in an 1:3 dilution in ddH2O (nuclease free).

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

For R Backbone (1, 2, 3):

Amplification of backbone (P)

Primers C1/C2 (071/072): (6700 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 70 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

DH5α colony picking

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with W, J, R, O, R4, E1, W3, M3 from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

ELISA

DH5α transformation & plating of constructs W, J, R, O, R4, E1, W3, M3

Goal

Transformation was performed to introduce plasmid DNA construct(s) … into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Agarose gel electrophoresis J, W, R, O

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: J,R,W and O. The first row shows the backbone samples while the second portrays the inserts. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples except for R (bb).

DpnI digest of constructs W, O, R, J

Goal

Restriction enzyme digest with DpnI was performed to cleave methylated plasmid DNA of the sample(s) …

Procedure

Digest was carried out using the DpnI restriction enzyme (New England Biolabs) according to the manufacturer’s instructions. 1 μg of DNA was combined with the 5 μL of 10X rCutSmart Buffer (New England Biolabs) in a 1.5 mL reaction vessel. Following the addition of 20 units of the restriction enzyme, the reaction mix was incubated at 37 °C for 60 min (ThermoMixer F1.5; Eppendorf). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
DNA	1 μg / … μL
10X rCutSmart Buffer	5 μL
DpnI	1 μL
ddH2O	… μL
Total	50 μL

Finally, the enzyme was heat-inactivated at 80 °C for 20 min (ThermoMixer F1.5; Eppendorf).

Results

The efficiency of the digest reaction was evaluated by subsequent experiments.

Gibson assembly of W, O, R, J

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into …

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
Master mix	5 μL
Vector	… ng / … μL
Insert	… ng / … μL
ddH2O	… μL
Total	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Q5 PCR for constructs J, O, R, W

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out without the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	100 ng / 1 μL
ddH2O	19 μL
Total	25 μL

In the end 1 μL polymerase was added in an 1:3 dilution in ddH2O (nuclease free).

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

For J:

Amplification of backbone (I)

Primers D1/J2 (081/141): (8000 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplification of insert (D)

Primers: J3/D4 (084/144): (800 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	01:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

For O:

Amplification of backbone (001, Guselkumab)

Primers E2/O1 (092/191): (6700 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplification of insert (N)

Primers: E3/O4 (093/192): (800 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	01:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

For R:

Amplification of backbone (P)

Primers C1/C2 (071/072): (6700 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplification of insert (Q)

Primers: C3/C4 (073/074): (1500 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	01:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

For W:

Amplification of backbone (U)

Primers C1/C2 (071/072): (6700 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	04:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplification of insert (V)

Primers: C3/C4 (073/074): (1500 bp)

Step	Condition
Initial denaturation	00:30, 98 °C
Denaturation	00:10, 98 °C
Annealing	00:30, 68 °C
Extension	01:00, 72 °C
Final extension	02:00, 72 °C
Cycles	×30

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Sanger sequencing of construct E, M, O, R

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of E, M, O, R using 46, 47, 64, 48 primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
HUJ628	46 fw	E1
HUJ629	64 rv	E1
HUJ630	46 fw	E2
HUJ631	64 rv	E2
HUJ632	46 fw	E3
HUJ633	64 rv	E3
HUJ634	46 fw	E4
HUJ562	64 rv	E4
08035623	46 fw	E5
08035624	64 rv	E5
06771834	46 fw	M2
06771835	64 rv	M2
06771836	46 fw	M3	success - > keep using
06771837	64 rv	M3
06771838	46 fw	M4
06771917	64 rv	M4
06771918	46 fw	M5
06771919	64 rv	M5
06771920	46 fw	O1
06771921	47 rv	O1
06771922	46 fw	O2
06771924	47 rv	O2
06771925	46 fw	O3
08035625	47 rv	O3
08035630	46 fw	R1
08035631	47 rv	R1
08035628	46 fw	R3
08035629	47 rv	R3
08035626	46 fw	R4	ree
08035627	47 rv	R4
08035632	46 fw	W2
08035633	47 rv	W2
08035634	46 fw	W3
08035635	47 rv	W3

Running 12% SDS Gel for Analysis of of Constructs 50/60, PQ, UV

Goal

Verification of the correct expression of the constructs from Expi293 via 12% SDS-Polyacrylamide gel.

Procedure

Fractions containing antibodies from FPLC are selected and reducing and non-reducing fractions are prepared for the sample application

reducing samples: 10ml 5 x Laemli Buffer (contains 10% ß-Mercaptoethanole) mixed with 40 µl of the FPLC fractions each

non-reducing samples: 10 ml 5 x NEM Laemli Buffer (contains 10% 1M NEM) mixed with 40 µl of the FPLC fraction each

Prepared samples are loaded according to the following loading scheme

Loading Bag

1

2

3

4

5

6

7

8

9

10

11

12

13

Gel 1 (Construct 50/60)

marker

supernatant

pooled fractions

Fraction 19 (red.)

Fraction 20 (red.)

Fraction 21 (red.)

Fraction 22 (red.)

free

free

free

Fraction 19 (non-red.)

Fraction 20 (non-red.)

Fraction 21 (non-red.)

Gel 2 (Construct PQ)

marker

supernatant

pooled fractions

Fraction 50 (red.)

Fraction 51 (red.)

Fraction 52 (red.)

Fraction 53 (red.)

free

free

free

Fraction 50 (non-red.)

Fraction 51 (non-red.)

Fraction 52 (non-red.)

Gel 3 (Construct UV)

marker

supernatant

pooled fractions

Fraction 81 (red.)

Fraction 82 (red.)

Fraction 83 (red.)

Fraction 84 (red.)

free

free

free

Fraction 81 (non-red.)

Fraction 82 (non-red.)

Fraction 83 (non-red.)

Pooled fractions were obtained by application of wash fractions of the FPLC. Every fraction was additionally denatured by cooking the diluted samples (Laemli + fraction) at 93 degrees Celsius prior to the application on the gel.

Results

Running 12% SDS Gel for Analysis of of Constructs 50/60, PQ und UV

Goal

Verification of the correct expression of the constructs from Expi293 via 12% SDS-Polyacrylamide gel.

Procedure

Fractions containing antibodies from FPLC are selected and reducing and non-reducing fractions are prepared for the sample application

reducing samples: 10ml 5 x Laemli Buffer (contains 10% ß-Mercaptoethanole) mixed with 40 µl of the FPLC fractions each

non-reducing samples: 10 ml 5 x NEM Laemli Buffer (contains 10% 1M NEM) mixed with 40 µl of the FPLC fraction each

Prepared samples are loaded according to the following loading scheme

Loading Bag	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Gel 1 (Construct 50/60)	marker	supernatant	pooled
Gel 2 (Construct UV)	marker
Gel 2 (Construct PQ)	marker	1+2 (21)	1+2 (22)	70 (52)	70 (53)	160 (54)	160 (55)	160 (56)	160 (57)

Pooled fractions were obtained by application of wash fractions of the FPLC. Every fraction was additionally denatured by cooking the diluted samples (Laemli + fraction) at 93 degrees celsius prior to the application on the gel.

Results

According to the developed gel the following fractions were selected and dialysed:

Determination of Antibody Concentration (constructs: K,I,F,50/60,P/Q,U/V)

In order to calculate the concentration via the Nanodrop machine, the molecular weight and estimated extinction coefficient were determined prior via the ExpasyProtparam Tool.

Results

Construct (FPLC fraction)	Concentration (mg/ml)
50/60	0,2194
P/Q	0,0707
U/V	0,1876
K (23)	0,4967
K (24)	1,3497
K (25)	0,3474
I (52)	0,0216
I (53)	0,134
I (54)	0,041
F (19)	0,1077
F (20)	0,84
F (21)	1,5574
F (22)	0,3312

Running 12% SDS Gel for Analysis of of Constructs K, I, F and N 1+2

Goal

Verification of the correct expression of the constructs from Expi293 via 12% SDS-Polyacrylamide gel.

Procedure

Fractions containing antibodies from FPLC are selected and reducing and non-reducing fractions are prepared for the sample application

reducing samples: 10ml 5 x Laemli Buffer (contains 10% ß-Mercaptoethanole) mixed with 40 µl of the FPLC fractions each

non-reducing samples: 10 ml 5 x NEM Laemli Buffer (contains 10% 1M NEM) mixed with 40 µl of the FPLC fraction each

Prepared samples are loaded according to the following loading scheme

Loading Bag

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Gel 1 (completely non-reducing samples + pooled wash fractions)

marker

K23

K24

K25

F19

F20

F21

I52

I53

I54

N54

N55

N56

pooled N38/39

pooled I35/36

Gel 2 (completely reducing samples)

marker

K23

K24

K25

F19

F20

F21

I52

I53

I54

N54

N55

N56

pooled K31/38 (non-reducing)

pooled F4/5 (non-reducing)

Pooled fractions were obtained by application of wash fractions of the FPLC. Every fraction was additionally denatured by cooking the diluted samples (Laemli + fraction) at 93 degrees celsius prior to the application on the gel.

Results

Gels were analyzed with the GelAnalyzer Software (the files are attached below)

Sanger sequencing of W, O, R, J, E, M I

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
	47 rev	W1
	48 fw	W1
	47 rev	W2
	48 fw	W2
	47 rev	W3
	48 fw	W3
	47 rev	W4
	48 fw	W4
	47 rev	W5
	48 fw	W5
	47 rev	O1
	48 fw	O1
	47 rev	O2
	48 fw	O2
	47 rev	O3
	48 fw	O3
	47 rev	O4
	48 fw	O4
	47 rev	O5
	48 fw	O5
	47 rev	R1
	48 fw	R1
	47 rev	R2
	48 fw	R2
	47 rev	R3
	48 fw	R3
	47 rev	R4
	48 fw	R4
	47 rev	R5
	48 fw	R5
	47 rev	J1
	48 fw	J1
	47 rev	J2
	48 fw	J2
	47 rev	J3
	48 fw	J3
	47 rev	J4
	48 fw	J4
	47 rev	J5
	48 fw	J5
	47 rev	E1
	48 fw	E1
	47 rev	E2
	48 fw	E2
	47 rev	E3
	48 fw	E3
	47 rev	E4
	48 fw	E4
	47 rev	E5
	48 fw	E5
	47 rev	M1
	48 fw	M1
	47 rev	M2
	48 fw	M2
	47 rev	M3
	48 fw	M3
	47 rev	M4
	48 fw	M4
	47 rev	M5
	48 fw	M5

Maxiprep of construct 30, 31, 30+H1, 30+K1 I

Goal

A maxiprep of 30, 31, 30+H1, 30+K1 was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)
30		1681.1
31		1511.3
30+H1		507
30+K1		1553.8

IDT sequencing primers Oligo resuspension of 46 47 48

This protocol is for resuspending IDT oligos.

Sanger sequencing of W, O, R, J, E, M

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
	47 rev	W1
	48 fw	W1
	47 rev	W2
	48 fw	W2
	47 rev	W3
	48 fw	W3
	47 rev	W4
	48 fw	W4
	47 rev	W5
	48 fw	W5
	47 rev	O1
	48 fw	O1
	47 rev	O2
	48 fw	O2
	47 rev	O3
	48 fw	O3
	47 rev	O4
	48 fw	O4
	47 rev	O5
	48 fw	O5
	47 rev	R1
	48 fw	R1
	47 rev	R2
	48 fw	R2
	47 rev	R3
	48 fw	R3
	47 rev	R4
	48 fw	R4
	47 rev	R5
	48 fw	R5
	47 rev	J1
	48 fw	J1
	47 rev	J2
	48 fw	J2
	47 rev	J3
	48 fw	J3
	47 rev	J4
	48 fw	J4
	47 rev	J5
	48 fw	J5
	47 rev	E1
	48 fw	E1
	47 rev	E2
	48 fw	E2
	47 rev	E3
	48 fw	E3
	47 rev	E4
	48 fw	E4
	47 rev	E5
	48 fw	E5
	47 rev	M1
	48 fw	M1
	47 rev	M2
	48 fw	M2
	47 rev	M3
	48 fw	M3
	47 rev	M4
	48 fw	M4
	47 rev	M5
	48 fw	M5

Miniprep of constructs 001, 299, 499

Goal

A miniprep of 001, 299, 499 was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)

Running 12% SDS Gel for Analysis of of Constructs 1+2, 70, 80, 150, 160, 180

Goal

Verification of the correct expression of the constructs from Expi293 via 12% SDS-Polyacrylamide gel.

Procedure

Fractions containing antibodies from FPLC are selected and reducing and non-reducing fractions are prepared for the sample application

reducing samples: 10ml 5 x Laemli Buffer (contains 10% ß-Mercaptoethanole) mixed with 40 µl of the FPLC fractions each

non-reducing samples: 10 ml 5 x NEM Laemli Buffer (contains 10% 1M NEM) mixed with 40 µl of the FPLC fraction each

Prepared samples are loaded according to the following loading scheme

Loading Bag	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
Gel 1 (completely non-reducing samples + pooled wash fractions)	marker	1+2 (21)	1+2 (22)	70 (52)	70 (53)	160 (54)	160 (55)	160 (56)	160 (57)	pooled fractions (Construct 70)	pooled fractions (Construct 80)	pooled fractions (Construct 150)	pooled fractions (Construct 160)	pooled fractions (Construct 180)	pooled fractions (Construct 1+2)
Gel 2 (completely reducing samples)	marker	1+2 (21)	1+2 (22)	70 (52)	70 (53)	160 (54)	160 (55)	160 (56)	160 (57)

Pooled fractions were obtained by application of wash fractions of the FPLC. Every fraction was additionally denatured by cooking the diluted samples (Laemli + fraction) at 93 degrees celsius prior to the application on the gel.

Results

The gel was analyzed with the GelAnalyzer Software (files are attached below)

DH5α transformation & suspension culture

Goal

Transformation was performed to introduce plasmid DNA construct(s) … into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 200 - 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

DH5α colony picking

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with … from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Antibody purification FPLC

Protocol for harvesting & purifying supernatant after recombinant/transient protein expression (antibodies) in Expi293 cells.

Running 12% SDS Gel for Analysis of of Constructs 50/60, UV and P+Q

Expi293F transfection of constructs N+2, F, I, D & K

Constructs … were transfected into a cell culture of Expi293 cells.

This experiment was performed according to the ThermoFishExpi293TM Expression System USER GUIDE.

This experiment is to be performed under sterile bench!

Explaination for the cell counter:

- Unpack cell counting chamber- 2x 10 μL TrypBlue onto packaging of cell counting chamber/eppi- around 20 μL form cells onto foil- 10 μL into each TrypBlue drop- Put 10 μL of the mixture into chamber A and B- Write down cell count

Running 12% SDS Gel for Analysis of of Constructs

Resequencing sanger sequencing R1-5, J1-5, G2, G3, H4

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of R1-5, J1-5, G2, G3, H4 using 47, 48, 64 primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
	47 rev	R1
	48 fw	R1
	47 rev	R2
	48 fw	R2
	47 rev	R2
	48 fw	R2
	47 rev	J1
	48 fw	J1
	47 rev	J2
	48 fw	J2
	47 rev	J2
	48 fw	J2
	47 rev	J3
	48 fw	J3
	47 rev	H4
	48 fw	H4
	47 rev	R3
	48 fw	R3
	47 rev	R4
	48 fw	R4
	47 rev	R5
	48 fw	R5
	47 rev	J4
	48 fw	J4
	47 rev	J5
	48 fw	J5
	47 rev	G2
	64 fw	G2
	47 rev	G3
	64 fw	G3
	47 rev	G5
	64 fw	G5

IDT primers Oligo resuspension

This protocol is for resuspending IDT oligos.

Q5 PCR of 001, 299, 499

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments using primers 301, 302 and 303, 304 for construct(s) 001, 299, 499 with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
Master mix	25 μL
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	1 μL
ddH2O	19 μL
Total	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	001 (primers 301/302)	299, 499 (primers 303/304)
Initial denaturation	3:00, 98 °C	3:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 70 °C	1:00, 68 °C
Extension	6:00, 72 °C	1:30, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments (c.f. attached file).

FPLC purification of 1+2, 70, 80, 150, 160, 180

Only purification of 1+2, 70 and 160 was successful. Expression of 80, 150 and 180 has to be repeated.

For protein purification from cell supernatant use FPLC purification protocol for Protein A Agarose column from iba lifesciences: https://www.iba-lifesciences.com/media/33/4a/54/1626347891/Manual_Protein_A_Agarose.pdf

1. for usage, the whole pump system has to be washed with MilliQ water (insert loop A, B and sample loop into water bottle) (manually)
2. then, wash the system with Buffer A (manually)
3. connect the column to the system (during slow Buffer A flow (0.5 mL/min) to prevent air bubble formation) (manually)
4. If Protein A Column is connected to the system, wash column again with Buffer A (25 mL, 5 CVs) (manually)
5. insert sample loop into the sample, make sure, that it reaches to the bottom of the falcon (to prevent air flow on the column) (manually)
6. Start program 20230817-Protein-A-5mL on the computer
7. Sample is applied to column, washed and eluted during the program (automatically)
8. Apply the following samples onto an SDS-PAGE to check if antibodies were correctly produced: supernatant (1x), pooling of washing fraction (1x), elution fractions (~ 4x) treated with beta-Mercaptoethanol for disulfide bond reduction, leave 3x pockets free, elution fractions treated non-reducing (NEM)

Detailed steps of the program down below:

Antibody purification FPLC of 1+2, 70, 80, 150, 160, 180

Protocol for harvesting & purifying supernatant after recombinant/transient protein expression (antibodies) in Expi293 cells.

Only purification of 1+2, 70 and 160 was successful. Expression of 80, 150 and 180 has to be repeated.

Dialysis

## Miniprep of construct W, O, R, J, E

Goal

A miniprep of W, O, P, J, E was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
E	1	750
	2	576
	3	609
	4	626
	5	513
J	1	403
	2	523
	3	914
	4	540
	5	606
O	1	623
	2	328
	3	239
	4	981
R	1	676
	2	408
	3	439
	4	836
	5	582
W	1	520
	2	860
	3	580
	4	793
	5	601

DH5α colony picking of W, O, R, E, J

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Sanger sequencing of D, E2, F, H3, K, O1

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of M and W using 47 (fw) 48 (rv) primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06771904	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771905	048_PCMV_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771906	064_B4_R_AR301-VH_IgG1-CH	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771907	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06771908	064_B4_R_AR301-VH_IgG1-CH	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06771909	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC
06771910	064_B4_R_AR301-VH_IgG1-CH	100_F_AR301-LC-57aaGS-HC
06771911	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 3
06771912	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 3
06771913	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
06771914	048_PCMV_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
06771915	047_NB_092_rev	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 1
06771916	048_PCMV_fw	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 1

Miniprep of construct W (5 times)

Goal

A miniprep of W was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
W1
W2
W3
W4
W5

Maxiprep of construct 30, 31, 30+H1, 30+K1

Goal

A maxiprep of 30, 31, 30+H1, 30+K1 was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)
30		1681.1
31		1511.3
30+H1		507
30+K1		1553.8

Sanger sequencing

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06771756	048_PCMV_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771757	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771758	064_B4_R_AR301-VH_IgG1-CH	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
06771759	048_PCMV_fw	100_F_AR301-LC-57aaGS-HC
06771760	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC
06771761	048_PCMV_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
06771762	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
06771763	048_PCMV_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC
06771764	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC
06771765	048_PCMV_fw	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 1
06771766	047_NB_092_rev	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 1
06771767	064_B4_R_AR301-VH_IgG1-CH	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 1
06771768	048_PCMV_fw	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 2
06771769	047_NB_092_rev	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 2
06771770	064_B4_R_AR301-VH_IgG1-CH	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 2
06771771	048_PCMV_fw	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 3
06771772	047_NB_092_rev	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 3
06771773	064_B4_R_AR301-VH_IgG1-CH	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 3
06771774	048_PCMV_fw	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 4
06771775	047_NB_092_rev	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 4
06771776	064_B4_R_AR301-VH_IgG1-CH	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 4
06771777	048_PCMV_fw	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 5
06771778	047_NB_092_rev	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 5
06771779	064_B4_R_AR301-VH_IgG1-CH	110_G_scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC / 5
06771780	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 1
06771781	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 1
06771782	064_B4_R_AR301-VH_IgG1-CH	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 1
06771783	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 2
06771784	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 2
06771785	064_B4_R_AR301-VH_IgG1-CH	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 2
06771786	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 3
06771787	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 3
06771788	064_B4_R_AR301-VH_IgG1-CH	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 3
06771789	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 4
06771790	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 4
06771791	064_B4_R_AR301-VH_IgG1-CH	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 4
06771792	048_PCMV_fw	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 5
06771793	047_NB_092_rev	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 5
06771794	064_B4_R_AR301-VH_IgG1-CH	120_H_AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC / 5
06771795	048_PCMV_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1
06771796	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1
06771797	048_PCMV_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06771798	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06771799	048_PCMV_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3
06771801	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3
06771802	048_PCMV_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 4
06771803	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 4
06771804	048_PCMV_fw	220_R_cG250-LC-2A-HC / 1
06771805	047_NB_092_rev	220_R_cG250-LC-2A-HC / 1
06771806	048_PCMV_fw	220_R_cG250-LC-2A-HC / 2
06771807	047_NB_092_rev	220_R_cG250-LC-2A-HC / 2
06771808	048_PCMV_fw	220_R_cG250-LC-2A-HC / 3
06771809	047_NB_092_rev	220_R_cG250-LC-2A-HC / 3
06771810	048_PCMV_fw	220_R_cG250-LC-2A-HC / 4
06771811	047_NB_092_rev	220_R_cG250-LC-2A-HC / 4
06771812	048_PCMV_fw	220_R_cG250-LC-2A-HC / 5
06771813	047_NB_092_rev	220_R_cG250-LC-2A-HC / 5
06771814	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 1
06771821	047_NB_092_rev	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 1
06771822	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 2
06771823	047_NB_092_rev	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 2
06771824	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 3
06771825	047_NB_092_rev	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 3
06771826	048_PCMV_fw	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 1
06771827	047_NB_092_rev	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 1
06771828	048_PCMV_fw	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 2
06771829	047_NB_092_rev	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 2
06771830	048_PCMV_fw	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 3
06771831	047_NB_092_rev	190_O_MOR4649-LC-11aaAPG-scFvhu128.1 / 3

Gibson assembly

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into …

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	E	G	H	J	M	O	R	W
Master mix	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL
Insert #1	49.21 ng / 1.3 μL	46.50 ng / 2 μL	31.54 ng / 0.5 μL	48.0 ng / 1.1 μL	—	56.0 ng / 1 μL	56.56 ng / 1 μL	54.66 ng / 1 μL
Insert #2	—	—	29.54 ng / 0.5 μL	—	—	—	—	—
ddH2O	2.7 μL	2 μL	3 μL	2.9 μL	4 μL	3 μL	3 μL	3 μL
Total	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

DpnI digest of 001, 299, 499

Goal

Restriction enzyme digest with DpnI was performed to cleave methylated plasmid DNA of the sample(s) …

Procedure

Digest was carried out using the DpnI restriction enzyme (New England Biolabs) according to the manufacturer’s instructions. 1 μg of DNA was combined with the 5 μL of 10X rCutSmart Buffer (New England Biolabs) in a 1.5 mL reaction vessel. Following the addition of 20 units of the restriction enzyme, the reaction mix was incubated at 37 °C for 60 min (ThermoMixer F1.5; Eppendorf). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
DNA	1 μg / … μL
10X rCutSmart Buffer	5 μL
DpnI	1 μL
ddH2O	… μL
Total	50 μL

Finally, the enzyme was heat-inactivated at 80 °C for 20 min (ThermoMixer F1.5; Eppendorf).

Results

The efficiency of the digest reaction was evaluated by subsequent experiments.

Q5 PCR

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
Master mix	25 μL
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	… ng / 1 μL
ddH2O	19 μL
Total	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	E-bb	E-i, H-i, J-i, O-i	G-bb, J-bb, M	G-i	H-bb	H-ii, R-i, W-i	O-bb	R-bb, W-bb
Initial denaturation	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 69 °C	1:00, 68 °C	1:00, 70 °C	1:00, 70 °C	1:00, 71 °C	1:00, 69 °C	1:00, 72 °C	1:00, 67 °C
Extension	6:00, 72 °C	1:30, 72 °C	6:00, 72 °C	1:30, 72 °C	6:00, 72 °C	1:30, 72 °C	6:00, 72 °C	6:00, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34	×34	×34	×34	×34	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Sanger sequencing

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
HUJ920	048_PCMV_fw	070_C_AR301-LC-2A-HC
HUJ921	047_NB_092_rev	070_C_AR301-LC-2A-HC
HUJ922	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC
HUJ923	048_PCMV_fw	070_C’_AR301-LC-2A-HC
HUJ924	047_NB_092_rev	070_C’_AR301-LC-2A-HC
HUJ925	064_B4_R_AR301-VH_IgG1-CH	070_C’_AR301-LC-2A-HC
HUJ926	048_PCMV_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
HUJ927	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
HUJ928	064_B4_R_AR301-VH_IgG1-CH	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC
HUJ929	048_PCMV_fw	100_F_AR301-LC-57aaGS-HC
HUJ930	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC
HUJ931	064_B4_R_AR301-VH_IgG1-CH	100_F_AR301-LC-57aaGS-HC
HUJ932	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
HUJ933	048_PCMV_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC
HUJ934	047_NB_092_rev	150_K’_MTfp-(G4S)2-AR301-LC-2A-HC
HUJ935	048_PCMV_fw	150_K’_MTfp-(G4S)2-AR301-LC-2A-HC
HUJ936	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC
HUJ937	048_PCMV_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC
HUJ938	047_NB_092_rev	160_L’_AR301-LC-(G4S)2-MTfp-2A-HC
HUJ939	048_PCMV_fw	160_L’_AR301-LC-(G4S)2-MTfp-2A-HC
HUJ940	047_NB_092_rev	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
HUJ941	048_PCMV_fw	180_N_scFvhu128.1-11aaAPG-MOR4649-LC

Sanger sequencing

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06768885	043_LKO.1 5’	700_Y_H1_pX458-sgRNA / 1
06768886	043_LKO.1 5’	700_Y_H1_pX458-sgRNA / 2
06768887	043_LKO.1 5’	700_Y_H1_pX458-sgRNA / 3
06768888	043_LKO.1 5’	700_Y_H1_pX458-sgRNA / 4
06768889	043_LKO.1 5’	700_Y_H1_pX458-sgRNA / 5
06768890	043_LKO.1 5’	710_Y_H2_pX458-sgRNA / 1
06768891	043_LKO.1 5’	710_Y_H2_pX458-sgRNA / 2
06768892	043_LKO.1 5’	710_Y_H2_pX458-sgRNA / 3
06768893	043_LKO.1 5’	710_Y_H2_pX458-sgRNA / 4
06768894	043_LKO.1 5’	720_Y_H3_pX458-sgRNA / 1
06768895	043_LKO.1 5’	720_Y_H3_pX458-sgRNA / 2
06768896	043_LKO.1 5’	720_Y_H3_pX458-sgRNA / 3
06768897	043_LKO.1 5’	720_Y_H3_pX458-sgRNA / 4
06768898	043_LKO.1 5’	720_Y_H3_pX458-sgRNA / 5
06768899	043_LKO.1 5’	750_Y_K1_pX458-sgRNA / 1
06768900	043_LKO.1 5’	750_Y_K1_pX458-sgRNA / 2
06768901	043_LKO.1 5’	750_Y_K1_pX458-sgRNA / 3
06768902	043_LKO.1 5’	750_Y_K1_pX458-sgRNA / 4
06768903	043_LKO.1 5’	750_Y_K1_pX458-sgRNA / 5
06768904	043_LKO.1 5’	760_Y_K2_pX458-sgRNA / 1
06768905	043_LKO.1 5’	760_Y_K2_pX458-sgRNA / 2
06768906	043_LKO.1 5’	760_Y_K2_pX458-sgRNA / 3
06768907	043_LKO.1 5’	760_Y_K2_pX458-sgRNA / 4
06768908	043_LKO.1 5’	760_Y_K2_pX458-sgRNA / 5
06768909	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 1
06768910	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 2
06768911	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 3
06768912	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 4
06768913	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 5
06768914	043_LKO.1 5’	770_Y_K3_pX458-sgRNA / 6
06768915	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1
06768916	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2
06768917	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3
06768918	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 4
06768919	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 5
06768920	046_NB_091_fw	100_F_AR301-LC-57aaGS-HC / 1
06768921	046_NB_091_fw	100_F_AR301-LC-57aaGS-HC / 2
06768922	046_NB_091_fw	100_F_AR301-LC-57aaGS-HC / 3
06768923	046_NB_091_fw	100_F_AR301-LC-57aaGS-HC / 4
06768924	046_NB_091_fw	100_F_AR301-LC-57aaGS-HC / 5
06768925	046_NB_091_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 1
06768926	046_NB_091_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 2
06768927	046_NB_091_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 3
06768928	046_NB_091_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 4
06768929	046_NB_091_fw	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 5
06768930	046_NB_091_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 1
06768931	046_NB_091_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 2
06768932	046_NB_091_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 3
06768933	046_NB_091_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 4
06768934	046_NB_091_fw	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 5
06768935	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1
06768936	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2
06768937	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3
06768938	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 4
06768939	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 5
06768940	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC / 1
06768941	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC / 2
06768942	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC / 3
06768943	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC / 4
06768944	047_NB_092_rev	100_F_AR301-LC-57aaGS-HC / 5
06768945	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 1
06768946	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 2
06768947	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 3
06768948	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 4
06768949	047_NB_092_rev	150_K_MTfp-(G4S)2-AR301-LC-2A-HC / 5
06768950	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 1
06768951	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 2
06768952	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 3
05682555	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 4
05682556	047_NB_092_rev	160_L_AR301-LC-(G4S)2-MTfp-2A-HC / 5

Q5 PCR

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
Master mix	25 μL
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	… ng / … μL
ddH2O	… μL
Total	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	…:…, 98 °C
Denaturation	…:…, 98 °C
Annealing	…:…, … °C
Extension	…:…, 72 °C
Final extension	…:…, 72 °C
Cycles	×…

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Gibson assembly

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into …

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	D	E	F	K	L	O	W
Master mix	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL
Insert	48 ng / 0.7 μL	48 ng / 0.6 μL	10 ng / 1 μL	-	-	60 ng / 0.6 μL	70 ng / 0.7 μL
ddH2O	3.3 μL	3.4 μL	3 μL	4 μL	4 μL	3.4 μL	3.3 μL
Total	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Sanger sequencing of C & N

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06768848	046_NB_091_fw	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
06768853	047_NB_092_rev	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
06768849	046_NB_091_fw	070_C_AR301-LC-2A-HC
06768854	047_NB_092_rev	070_C_AR301-LC-2A-HC
06768850	046_NB_091_fw	070_C’_AR301-LC-2A-HC
06768855	047_NB_092_rev	070_C’_AR301-LC-2A-HC

DpnI digest

Goal

Restriction enzyme digest with DpnI was performed to cleave methylated plasmid DNA of the sample(s) …

Procedure

Digest was carried out using the DpnI restriction enzyme (New England Biolabs) according to the manufacturer’s instructions. 1 μg of DNA was combined with the 5 μL of 10X rCutSmart Buffer (New England Biolabs) in a 1.5 mL reaction vessel. Following the addition of 20 units of the restriction enzyme, the reaction mix was incubated at 37 °C for 60 min (ThermoMixer F1.5; Eppendorf). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
DNA	1 μg / … μL
10X rCutSmart Buffer	5 μL
DpnI	1 μL
ddH2O	… μL
Total	50 μL

Finally, the enzyme was heat-inactivated at 80 °C for 20 min (ThermoMixer F1.5; Eppendorf).

Results

The efficiency of the digest reaction was evaluated by subsequent experiments.

Gibson assembly I

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into …

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	D	E	F
Master mix	5 μL	5 μL	5 μL
Vector	98.01 ng / 1 μL	99.14 ng / 1 μL	86.79 ng / 1 μL
Insert	42.84 ng / 0.5 μL	49.76 ng / 0.5 μL	48.26 ng / 0.5 μL
ddH2O	3.5 μL	3.5 μL	3.5 μL
Total	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Miniprep

Goal

A miniprep of … was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)

Q5 PCR

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	D-bb	D-i	E-bb	E-i	F-bb	F-i
Master mix	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	10 ng / 1 μL	100 ng / 1 μL	10 ng / 1 μL	100 ng / 1 μL	10 ng / 1 μL
ddH2O	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL
Total	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	D-bb	D-i	E-bb	E-i	F-bb	F-i
Initial denaturation	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C	3:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 69 - 71 °C	1:00, 70 °C	1:00, 69 - 71 °C	1:00, 68 °C	1:00, 69 - 71 °C	1:00, 70 °C
Extension	6:00, 72 °C	6:00, 72 °C	6:00, 72 °C	6:00, 72 °C	6:00, 72 °C	6:00, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34	×34	×34	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Gibson assembly

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into …

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	O	W
Master mix	5 μL	5 μL
Vector	86.79 ng / 1 μL	101.33 ng / 1 μL
Insert	48.26 ng / 0.5 μL	114.61 ng / 1.3 μL
ddH2O	3.5 μL	2.7 μL
Total	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Q5 PCR for O, W (Redo)

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from … using primers … for construct(s) … with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	O-bb	O-i	W-bb	W-i
Master mix	25 μL	25 μL	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	2 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL
ddH2O	19 μL	19 μL	19 μL	19 μL
Total	50 μL	50 μL	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	O-bb	O-i	W-bb	W-i
Initial denaturation	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 72 °C	1:00, 69 °C	1:00, 67 °C	1:00, 69 °C
Extension	5:00, 72 °C	1:30, 72 °C	5:00, 72 °C	1:30, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Miniprep of C-SDM (with DpnI digest)

Goal

A miniprep of C (AR301-LC-2A-HC) was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	6	428.30
070_C_AR301-LC-2A-HC	7	474.15
070_C_AR301-LC-2A-HC	8	476.25
070_C_AR301-LC-2A-HC	9	478.50
070_C_AR301-LC-2A-HC	10	365.70
070_C_AR301-LC-2A-HC	11	481.60
070_C_AR301-LC-2A-HC	12	541.65

Sanger sequencing of C-SDM, N, R

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06768797	046_NB_091_fw	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
06768798	047_NB_092_rev	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
06768799	054_A4_R_AR301-VL_IgL2_CL	180_N_scFvhu128.1-11aaAPG-MOR4649-LC
06768800	046_NB_091_fw	220_R_cG250-LC-2A-HC
06768801	047_NB_092_rev	220_R_cG250-LC-2A-HC
06768802	064_B4_R_AR301-VH_IgG1-CH	220_R_cG250-LC-2A-HC
06768808	046_NB_091_fw	070_C_AR301-LC-2A-HC / 1
06768796	047_NB_092_rev	070_C_AR301-LC-2A-HC / 1
06768809	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 1
06768803	046_NB_091_fw	070_C_AR301-LC-2A-HC / 2
06768804	047_NB_092_rev	070_C_AR301-LC-2A-HC / 2
06768812	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 2
06768805	046_NB_091_fw	070_C_AR301-LC-2A-HC / 3
06764132	047_NB_092_rev	070_C_AR301-LC-2A-HC / 3
06768815	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 3
06764135	046_NB_091_fw	070_C_AR301-LC-2A-HC / 4
06768845	047_NB_092_rev	070_C_AR301-LC-2A-HC / 4
06768818	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 4
06768846	046_NB_091_fw	070_C_AR301-LC-2A-HC / 5
06768847	047_NB_092_rev	070_C_AR301-LC-2A-HC / 5
06768821	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 5
06768824	046_NB_091_fw	070_C_AR301-LC-2A-HC / 6
06768825	047_NB_092_rev	070_C_AR301-LC-2A-HC / 6
06768826	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 6
06768827	046_NB_091_fw	070_C_AR301-LC-2A-HC / 7
06768828	047_NB_092_rev	070_C_AR301-LC-2A-HC / 7
06768829	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 7
06768830	046_NB_091_fw	070_C_AR301-LC-2A-HC / 8
06768831	047_NB_092_rev	070_C_AR301-LC-2A-HC / 8
06768832	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 8
06768833	046_NB_091_fw	070_C_AR301-LC-2A-HC / 9
06768834	047_NB_092_rev	070_C_AR301-LC-2A-HC / 9
06768835	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 9
06768836	046_NB_091_fw	070_C_AR301-LC-2A-HC / 10
06768837	047_NB_092_rev	070_C_AR301-LC-2A-HC / 10
06768838	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 10
06768839	046_NB_091_fw	070_C_AR301-LC-2A-HC / 11
06768840	047_NB_092_rev	070_C_AR301-LC-2A-HC / 11
06768841	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 11
06768842	046_NB_091_fw	070_C_AR301-LC-2A-HC / 12
06768843	047_NB_092_rev	070_C_AR301-LC-2A-HC / 12
06768844	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 12

Miniprep of C-SDM (w/o DpnI digest)

Goal

A miniprep of C (AR301-LC-2A-HC) was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	1	221.95
070_C_AR301-LC-2A-HC	2	279.75
070_C_AR301-LC-2A-HC	3	286.65
070_C_AR301-LC-2A-HC	4	246.95
070_C_AR301-LC-2A-HC	5	367.25

DH5α colony picking of C-SDM

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with C (AR301-LC-2A-HC) from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

DH5α transformation with C-SDM & plating

Goal

Transformation was performed to introduce plasmid DNA construct C (AR301-LC-2A-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

DpnI digest of C-SDM

Goal

Restriction enzyme digest with DpnI was performed to cleave methylated plasmid DNA of the PCR sample for constructs C (AR301-LC-2A-HC).

Procedure

Digest was carried out using the DpnI restriction enzyme (New England Biolabs) according to the manufacturer’s instructions. 1 μg of DNA was combined with the 5 μL of 10X rCutSmart Buffer (New England Biolabs) in a 1.5 mL reaction vessel. Following the addition of 20 units of the restriction enzyme, the reaction mix was incubated at 37 °C for 60 min (ThermoMixer F1.5; Eppendorf). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
DNA	1 μg / 4 μL
10X rCutSmart Buffer	5 μL
DpnI	1 μL
ddH2O	40 μL
Total	50 μL

Finally, the enzyme was heat-inactivated at 80 °C for 20 min (ThermoMixer F1.5; Eppendorf).

Results

The efficiency of the digest reaction was evaluated by subsequent experiments.

Gibson assembly of C-SDM

Goal

Gibson assembly was performed to assemble the mutagenised DNA fragments into construct C (AR301-LC-2A-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	C w/o DpnI digest	C with DpnI digest
Master mix	5 μL	5 μL
DNA	150 ng / 0.5 μL	100 ng / 1 μL
ddH2O	4.5 μL	4 μL
Total	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Q5 PCR for C-SDM

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragment from 070_C_AR301-LC-2A-HC using primers 077_C_F_SDM_2A and 078_C_R_SDM_2A for site-directed mutagenesis with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	Amount
Master mix	25 μL
10 μM forward primer	2.5 μL
10 μM reverse primer	2.5 μL
Template DNA	100 ng / 1 μL
ddH2O	19 μL
Total	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	2:00, 98 °C
Denaturation	0:30, 98 °C
Annealing	1:00, 66 °C
Extension	6:00, 72 °C
Final extension	10:00, 72 °C
Cycles	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Dialysis of Contructs 50/60, U+V and P+Q

Goal

Transfer of Antibodies from Elution Buffer from FPLC to PBS for storage

Materials

to be added (don’t know yet)

Running 12% SDS Gel for Analysis of of Constructs 50/60, UV and P+Q

Goal

Verification of the correct expression of the constructs from Expi293 via 12% SDS-Polyacrylamide gel.

Procedure

Fractions containing antibodies from FPLC are selected and reducing and non-reducing fractions are prepared for the sample application

reducing samples: 10ml 5 x Laemli Buffer (contains 10% ß-Mercaptoethanole) mixed with 40 µl of the FPLC fractions each

non-reducing samples: 10 ml 5 x NEM Laemli Buffer (contains 10% NEM) mixed with 40 µl of the FPLC fraction each

Prepared samples are loaded according to the following loading scheme

Laoding Bag

1

2

3

4

5

6

7

8

9

10

11

12

13

Gel 1 (Constructs 50/60)

marker

Supernatant

FT (fraction 3-7)

fraction 19 (red.)

fraction 20 (red.)

fraction 21 (red.)

fraction 22 (red.)

water + 1x laemli

water + 1 x laemli

water + 1x laemli

fraction 19 (non-red.)

Fraction 20 (non-red.)

fraction 21 (non-red.)

Gel 2 (Construct U+V)

marker

Supernatant

FT

fraction 50 (red.)

fraction 51 (red.)

fraction 52 (red.)

fraction 53 (red.)

freewater + 1x laemli

water + 1x laemli

water + 1x laemli

fraction 50 (non-red.)

fraction 51 (non-red.)

fraction 52 (non-red.)

Gel 3 (Cosntruct P+Q)

marker

Supernatant

FT (fractions 65-69)

fraction 81 (red.)

fraction 82 (red.)

fraction 83 (red.)

fraction 84 (red.)

water + 1x laemli

water + 1x laemli

water + 1x laemli

fraction 81 (non-red.)

fraction 82 (non-red.)

fraction 83 (non-red.)

The supernatant was contained by centrifugation of the cell culture. Every fraction was additionally denatured by cooking the diluted samples (Laemli + fraction) at 93 degrees celsius prior to the application on the gel.

Results

According to the developed gel the following fractions were selected and dialysed:

Gel 1 (50/60): 19-21

Gel 2 (U+V): 50-52

Gel 3 (P+Q): 81-84

FPLC purification - constructs 50-60, U-V and P-Q

For protein purification from cell supernatant use FPLC purification protocol for Protein A Agarose column from iba lifesciences: https://www.iba-lifesciences.com/media/33/4a/54/1626347891/Manual_Protein_A_Agarose.pdf

1. for usage, the whole pump system has to be washed with MilliQ water (insert loop A, B and sample loop into water bottle) (manually)
2. then, wash the system with Buffer A (manually)
3. connect the column to the system (during slow Buffer A flow (0.5 mL/min) to prevent air bubble formation) (manually)
4. If Protein A Column is connected to the system, wash column again with Buffer A (25 mL, 5 CVs) (manually)
5. insert sample loop into the sample, make sure, that it reaches to the bottom of the falcon (to prevent air flow on the column) (manually)
6. Start program 20230817-Protein-A-5mL on the computer
7. Sample is applied to column, washed and eluted during the program (automatically)
8. Apply the following samples onto an SDS-PAGE to check if antibodies were correctly produced: supernatant (1x), pooling of washing fraction (1x), elution fractions (~ 4x) treated with beta-Mercaptoethanol for disulfide bond reduction, leave 3x pockets free, elution fractions treated non-reducing (NEM)

Detailed steps of the program down below:

Result: Purification was successful, see SDS-PAGE images of presumable antibodies!

Harvesting cells for FPLC purification - constructs 50-60, U-V and P-Q

Protocol for harvesting & purifying supernatant after recombinant/transient protein expression (antibodies) in Expi293 cells.

Preparations:

• cool down centrifuge
• during centrifugation steps, start preparing the column and equilibrating the FPLC

Sanger sequencing of Q

Goal

DNA sequencing was performed to confirm the nucleotide sequence(s) of … using … primer(s).

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation

Maxiprep of Q

Goal

A maxiprep of Q was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)

Expi293F transfection with constructs PQ, 50 60, UV

Constructs PQ, UV and 50 60 were transfected into a cell culture of Expi293 cells.

This experiment was performed according to the ThermoFishExpi293TM Expression System USER GUIDE. The instructions are to be performed for each sample individually.

This experiment is to be performed under sterile bench!

Explaination for the cell counter:

- Unpack cell counting chamber- 2x 10 μL TrypBlue onto packaging of cell counting chamber/eppi- around 20 μL form cells onto foil- 10 μL into each TrypBlue drop- Put 10 μL of the mixture into chamber A and B- Write down cell count

DH5α transformation & suspension culture

Goal

Transformation was performed to introduce plasmid DNA construct(s) … into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 200 - 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

DH5α colony picking of C, R, W, N, O

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with C (AR301-LC-2A-HC) from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Maxiprep of P, Q, U, V & J

Goal

A maxiprep of P (cG250-LC), Q (cG250-HC), U (006.11-LC), V (006.11-HC), and J (scFvhu128.1-11aaAPG-scFvAR301-HC) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)

DH5α transformation with C, R, W, N, O & plating

Goal

Transformation was performed to introduce plasmid DNA constructs C (AR301-LC-2A-HC), R (cG250-LC-2A-HC), W (006.11-LC-2A-HC), N (scFvhu128.1-11aaAPG-MOR4649-LC), and O (MOR4649-LC-11aaAPG-scFvhu128.1) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Gibson assembly of C, R, W, N & O

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into C (AR301-LC-2A-HC), R (cG250-LC-2A-HC), W (006.11-LC-2A-HC), N (scFvhu128.1-11aaAPG-MOR4649-LC), and O (MOR4649-LC-11aaAPG-scFvhu128.1).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	C (GelEx + AP)	R (GelEx + AP)	W (GelEx + AP)	C (AP)	R (AP)	W (AP)	N (AP)	O (AP)
Master mix	10 μL	10 μL	10 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	65 ng / 8 μL	65 ng / 5.4 μL	65 ng / 4.2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL
Insert	69 ng / 1.3 μL	69 ng / 1.9 μL	69 ng / 1.65 μL	107 ng / 2.15 μL	107 ng / 2.15 μL	107 ng / 2.15 μL	37 ng / 1.85 μL	37 ng / 1.85 μL
ddH2O	0.7 μL	2.7 μL	4.15 μL	0.85 μL	0.85 μL	0.85 μL	1.15 μL	1.15 μL
Total	20 μL	20 μL	20 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Gel extraction of C, R, W, N, O

Goal

A gel extraction of C (C_AR301-LC-2A-HC) was performed to isolate and purify a specific DNA fragment from agarose gel for downstream applications.

Procedure

Following gel electrophoresis, DNA bands of interest were excised from the gel under UV transillumination (SERVA) using a clean scalpel. The excised gel slices were transferred to a 1.5 mL microcentrifuge tube and weighed to determine the amount of gel present. DNA fragments were then isolated and purified from gel using the Wizard SV Gel and PCR Clean-Up System (Promega) following modified manufacturer’s instructions. For this, the gel slices were first dissolved in 10 μL of Membrane Binding Solution (Promega) per 10 mg of gel at 50 - 65 °C. The mixture was then transferred to an SV Minicolumn placed in a collection tube, and incubated for 1 min at room temperature. The column was centrifuged at 16000 × g or 13053 rpm (Centrifuge 5425; Eppendorf) for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed with 700 μL and subsequently again with 500 μL Membrane Wash Solution (Promega) supplemented with 95% ethanol (4:1) at 16000 × g for 1 min each to remove impurities and contaminants. Excess ethanol was removed by centrifuging the column for additional 5 min at 16000 × g. Purified PCR products were eluted into a 1.5 mL microcentrifuge tube by adding 25 μL of nuclease-free water to the column matrix, incubating at room temperature for 3 min, and centrifuging the column for 1 min at 16000 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The extraction procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Product	Concentration (ng/μL)
C bb	8.4
C insert	54.3
R bb	12.2
R insert	35.9
W bb	15.6
W insert	42.0
O bb	14.3
O insert	47.6
N bb	19.1
D,G,N insert	64.0
E insert	37.8
F linker	-
H insert	34.2
H linker	7.6

Agarose gel electrophoresis for C, R, W, D, E, F, G, H, N & O

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, backbone and insert PCR fragments for construct C (AR301-LC-57aaGS-HC), backbone and insert PCR fragments for construct R (cG250-LC-2A-HC), backbone and insert PCR fragments for construct W (006.11-LC-2A-HC), backbone and insert PCR fragments for construct O (MOR4649-LC-11aaAPG-scFvhu128.1), backbone PCR fragment for construct N (scFvhu128.1-11aaAPG-MOR4649-LC), empty lane, insert PCR fragment for constructs D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), G (scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC), and N, insert PCR fragment for construct E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), insert PCR fragment for construct F (AR301-LC-57aaGS-HC), first and second insert PCR fragments for construct H (AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC), ladder, ladder. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples. No desired product was, however, recorded for insert of the construct F.

Expi293 subculturing

Goal:

Expi293 cell splitting was performed to maintain the cells in an optimal growth state.

Procedure:

Expi293 cells (Product No. A14527; Gibco) were cultured to a density of 3 - 5 × 10⁶ cells/mL. Subculturing was performed by splitting the cells to the desired cell concentration of 0.3 × 10⁶ cells/mL using pre-warmed Expi293 Expression Medium (Gibco). The suspension culture was maintained at 37 °C in a humidified incubator (BINDER) with 8% CO₂. To ensure optimal performance, cell density was not allowed to exceed 5 × 10⁶ cells/mL, and cells were discarded after passage number 30. Cell viability and growth were monitored using an automatic cell counter (Countess 3; Invitrogen) and trypan blue exclusion staining (Countess cell counting chamber slides; Invitrogen).

Results:

Cells were successfully split at density of 4.24 × 10⁶ cells/mL and 99% viability.

Q5 PCR for R, W, D, E, F, G, H, N & O

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from 200_P_cG250-LC and 210_Q_cG250-HC using primers 071_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A and 073_C3_F_Leader(H)_2A, 074_C4_R_IgG1-CH_pcDNA respectively for construct R (cG250-LC-2A-HC), from 250_U_006.11-LC and 260_V_006.11-HC using primers 071_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A and 073_C3_F_Leader(H)_2A, 074_C4_R_IgG1-CH_pcDNA respectively for construct W (006.11-LC-2A-HC), from 009_scFv-hu128.1 with primers 083_D3_F_hu128.1-VL_Leader(L), 084_D4_R_hu128.1-VH_11aaAPG for constructs D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), G (scFvhu128.1-11aaAPG-AR301-LC-57aaGS-HC), from 001_MOR4649-LC and 009_scFv-hu128.1 with primers 181_N1_F_MOR4649-VL_11aaAPG, 082_D2_R_Leader(L)_hu128.1-VL and 083_D3_F_hu128.1-VL_Leader(L), 084_D4_R_hu128.1-VH_11aaAPG respectively for construct N (scFvhu128.1-11aaAPG-MOR4649-LC), from 009_scFv-hu128.1 with primers 093_E3_F_hu128.1-VL_11aaAPG, 094_E4_R_hu128.1-VH_2A for construct E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), from 009_scFv-hu128.1 and 022_108aa-GS-Linker with primers 093_E3_F_hu128.1-VL_11aaAPG, 122_H4_R_hu128.1-VH_108aaGS and 123_H5_F_108aaGS_hu128.1-VH, 124_H6_R_108aaGS_AR301-VH respectively for construct H (AR301-LC-11aaAPG-scFvhu128.1-108aaGS-HC), from 001_MOR4649-LC and 009_scFv-hu128.1 with primers 191_O1_F_pcDNA_hu128.1-VH, 092_E2_R_IgL2-CL_11aaAPG and 093_E3_F_hu128.1-VL_11aaAPG, 192_O4_R_hu128.1-VH_pcDNA respectively for construct O (MOR4649-LC-11aaAPG-scFvhu128.1), from 022_108aa-GS-Linker using primers 103_F3_F_57aaGS_IgL2-CL, 104_F4_R_57aaGS_AR301-VH for construct F (AR301-LC-57aaGS-HC) with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	R-i	R-bb	W-i	W-bb	D/G/N-i	N-bb	E-i	H-i1	H-i2	O-i	O-bb	F-i
Master mix	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	2 ng / 1 μL	100 ng / 1 μL	2 ng / 1 μL	2 ng / 1 μL	2 ng / 1 μL	2 ng / 1 μL	100 ng / 1 μL	2 ng / 1 μL
ddH2O	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL
Total	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	R-bb, W-bb, O-bb	N-bb	D/G/N-i, F-i	R-i, W-i, H-i2	E-i, H-i1, O-i
Initial denaturation	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 72 °C	1:00, 71 °C	1:00, 70 °C	1:00, 69 °C	1:00, 68 °C
Extension	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34	×34	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

DH5α transformation with P, Q, U, V, J & suspension culture

Goal

Transformation was performed to introduce plasmid DNA constructs P (cG250-LC), Q (cG250-HC), U (006.11-LC), V (006.11-HC), and J (scFvhu128.1-11aaAPG-scFvAR301-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 200 - 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Miniprep of C

Goal

A miniprep of C (AR301-LC-2A-HC) was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	4	713
070_C_AR301-LC-2A-HC	5	622
070_C_AR301-LC-2A-HC	6	674

Sanger sequencing of C

Goal

DNA sequencing was performed to confirm the nucleotide sequence of C (AR301-LC-2A-HC) using PCMV_fw and NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06768524	047_NB_092_rev	070_C_AR301-LC-2A-HC / 1	Failed insertion, religation of A
06768525	047_NB_092_rev	070_C_AR301-LC-2A-HC / 2	Failed insertion, religation of A
06768526	047_NB_092_rev	070_C_AR301-LC-2A-HC / 3	Failed insertion, religation of A
06768741	047_NB_092_rev	070_C_AR301-LC-2A-HC / 1’	Failed insertion, religation of A
06768742	047_NB_092_rev	070_C_AR301-LC-2A-HC / 2’	Failed insertion, religation of A
06768647	047_NB_092_rev	070_C_AR301-LC-2A-HC / 3’	Failed insertion, religation of B
06768648	048_PCMV_fw	070_C_AR301-LC-2A-HC / 4	Failed insertion, religation of A
06768649	047_NB_092_rev	070_C_AR301-LC-2A-HC / 4	Failed insertion, religation of A
06768650	048_PCMV_fw	070_C_AR301-LC-2A-HC / 5	Failed insertion, religation of A
06768651	047_NB_092_rev	070_C_AR301-LC-2A-HC / 5	Failed insertion, religation of A
06768652	048_PCMV_fw	070_C_AR301-LC-2A-HC / 6	Failed insertion, religation of A
06768653	047_NB_092_rev	070_C_AR301-LC-2A-HC / 6	Failed insertion, religation of A

DH5α colony picking of C

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with C (AR301-LC-2A-HC) from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Sanger sequencing of C, P, Q, U, V & J

Goal

DNA sequencing was performed to confirm the nucleotide sequences of C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), V (006.11-HC), and J (scFvhu128.1-11aaAPG-scFvAR301-HC) using PCMV_fw and B4_R_AR301-VH_IgG1-CH or 047_NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06768482	048_PCMV_fw	070_C_AR301-LC-2A-HC / 1	No insert, A religated
06768483	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 1	No insert, A religated
06768484	048_PCMV_fw	070_C_AR301-LC-2A-HC / 2	No insert, A religated
06768485	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 2	No insert, A religated
06768486	048_PCMV_fw	070_C_AR301-LC-2A-HC / 3	B religated
06768487	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 3	B religated
06768488	048_PCMV_fw	070_C_AR301-LC-2A-HC / 1’	No insert, A religated
06768489	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 1’	No insert, A religated
06768490	048_PCMV_fw	070_C_AR301-LC-2A-HC / 2’	No insert, A religated
06768491	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 2’	No insert, A religated
06768492	048_PCMV_fw	070_C_AR301-LC-2A-HC / 3’	No insert, A religated
06768493	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC / 3’	No insert, A religated
06768494	048_PCMV_fw	200_P_cG250-LC / 1	Confirmed
06768495	047_NB_092_rev	200_P_cG250-LC / 1	Confirmed
06768496	048_PCMV_fw	200_P_cG250-LC / 2	Confirmed
06768497	047_NB_092_rev	200_P_cG250-LC / 2	Confirmed
06768498	048_PCMV_fw	200_P_cG250-LC / 3	Confirmed
06768499	047_NB_092_rev	200_P_cG250-LC / 3	No priming
06768500	048_PCMV_fw	210_Q_cG250-HC / 1	Confirmed
06768501	047_NB_092_rev	210_Q_cG250-HC / 1	Confirmed
06768502	048_PCMV_fw	210_Q_cG250-HC / 2	No insert, backbone religated
06768503	047_NB_092_rev	210_Q_cG250-HC / 2	No insert, backbone religated
06768504	048_PCMV_fw	210_Q_cG250-HC / 3	No insert, backbone religated
06768505	047_NB_092_rev	210_Q_cG250-HC / 3	No insert, backbone religated
06768506	048_PCMV_fw	250_U_006.11-LC / 1	Confirmed
06768507	047_NB_092_rev	250_U_006.11-LC / 1	Confirmed
06768510	048_PCMV_fw	250_U_006.11-LC / 3	Confirmed
06768511	047_NB_092_rev	250_U_006.11-LC / 3	Confirmed
06768512	048_PCMV_fw	260_V_006.11-HC / 1	Confirmed
06768513	047_NB_092_rev	260_V_006.11-HC / 1	Confirmed
06768514	048_PCMV_fw	260_V_006.11-HC / 2	Confirmed
06768515	047_NB_092_rev	260_V_006.11-HC / 2	Confirmed
06768516	048_PCMV_fw	260_V_006.11-HC / 3	No insert, backbone religated
06768517	047_NB_092_rev	260_V_006.11-HC / 3	No insert, backbone religated
06768518	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 1	Possible mutation C1165T
06768519	064_B4_R_AR301-VH_IgG1-CH	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 1	Possible mutation C1165T
06768520	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 2	Several uncertainties
06768521	064_B4_R_AR301-VH_IgG1-CH	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 2	Several uncertainties
06768522	048_PCMV_fw	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 3	Confirmed
06768523	064_B4_R_AR301-VH_IgG1-CH	140_J_scFvhu128.1-11aaAPG-scFvAR301-HC / 3	Confirmed

Miniprep of C, P, Q, U, V & J

Goal

A miniprep of C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), V (006.11-HC), and J (scFvhu128.1-11aaAPG-scFvAR301-HC) was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	1	987
070_C_AR301-LC-2A-HC	2	546
070_C_AR301-LC-2A-HC	3	752
070_C_AR301-LC-2A-HC	1’	684
070_C_AR301-LC-2A-HC	2’	712
070_C_AR301-LC-2A-HC	3’	700
200_P_cG250-LC	1	408
200_P_cG250-LC	2	693
200_P_cG250-LC	3	558
210_Q_cG250-HC	1	786
210_Q_cG250-HC	2	722
210_Q_cG250-HC	3	651
250_U_006.11-LC	1	767
250_U_006.11-LC	2	-
250_U_006.11-LC	3	689
260_V_006.11-HC	1	507
260_V_006.11-HC	2	873
260_V_006.11-HC	3	577
140_J_scFvhu128.1-11aaAPG-scFvAR301-HC	1	581
140_J_scFvhu128.1-11aaAPG-scFvAR301-HC	2	966
140_J_scFvhu128.1-11aaAPG-scFvAR301-HC	3	521

DH5α transformation with C & plating (Redo)

Goal

Transformation was performed to introduce plasmid DNA constructs C (AR301-LC-2A-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

No or little to no colonies were present on the selective agar plate after overnight incubation.

Gibson assembly for C, P, Q, U & V (Redo)

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	C	C’	P	Q	U	V
Master mix	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL
Insert	107 ng / 2.15 μL	107 ng / 2.15 μL	27 ng / 1.35 μL	25 ng / 1.25 μL	27 ng / 1.35 μL	28.5 ng / 1.43 μL
ddH2O	0.85 μL	0.85 μL	1.65 μL	1.75 μL	1.65 μL	1.57 μL
Total	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Agarose gel electrophoresis of PCR fragments for C, P, Q, U & V

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, backbone PCR fragment for construct C (AR301-LC-57aaGS-HC), backbone PCR fragment for construct P (cG250-LC), backbone PCR fragment for construct Q (cG250-HC), backbone PCR fragment for construct U (006.11-LC), backbone PCR fragment for construct V (006.11-HC), empty, unrelated DNA sample. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in all samples.

DH5α colony picking of J, C, P, Q, U & V

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC), as well as J (scFvhu128.1-11aaAPG-scFvAR301-HC) from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

DH5α transformation with J, C, P, Q, U, V & plating

Goal

Transformation was performed to introduce plasmid DNA constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC), as well as J (scFvhu128.1-11aaAPG-scFvAR301-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

No or little to no colonies were present on the selective agar plate after overnight incubation.

Gibson assembly for C, P, Q, U & V (Redo)

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	C	C’	P	Q	U	V
Master mix	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL
Insert	107 ng / 2.15 μL	107 ng / 2.15 μL	27 ng / 1.35 μL	25 ng / 1.25 μL	27 ng / 1.35 μL	28.5 ng / 1.43 μL
ddH2O	0.85 μL	0.85 μL	1.65 μL	1.75 μL	1.65 μL	1.57 μL
Total	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Q5 PCR for C, P, Q, U & V (Redo)

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from 050_A_AR301-LC using primers 071_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A as well as primers 075_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A for construct C (AR301-LC-2A-HC), from 001_MOR4649-LC using primers 201_P1_F_IgL2-CL_cG250-VL, 202_P2_R_Leader(L)_cG250-VL for construct P (cG250-LC), from 002_MOR4649-HC using primers 211_Q1_F_IgG1-CH_cG250-VH, 212_Q2_R_Leader(H)_cG250-VH for construct Q (cG250-HC), from 001_MOR4649-LC using primers 251_U1_F_IgL2-CL_006.11-VL, 252_U2_R_Leader(L)_006.11-VL for construct U (006.11-LC), from 002_MOR4649-HC using primers 261_V1_F_IgG1-CH_006.11-VH, 262_V2_R_Leader(H)_006.11-VH for construct V (006.11-HC) with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	C	C’	P	Q	U	V
Master mix	25 μL	25 μL	25 μL	25 μL	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL	100 ng / 1 μL
ddH2O	19 μL	19 μL	19 μL	19 μL	19 μL	19 μL
Total	50 μL	50 μL	50 μL	50 μL	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	C	C’	P	Q	U	V
Initial denaturation	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C	2:00, 98 °C
Denaturation	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C	0:30, 98 °C
Annealing	1:00, 72 °C	1:00, 67 °C	1:00, 72 °C	1:00, 70 °C	1:00, 72 °C	1:00, 70 °C
Extension	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C	5:00, 72 °C
Final extension	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C	10:00, 72 °C
Cycles	×34	×34	×34	×34	×34	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Gibson assembly for construct J

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into construct J (scFvhu128.1-11aaAPG-scFvAR301-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
Master mix	5 μL
Vector	100 ng / 2 μL
Insert	47 ng / 2.15 μL
ddH2O	0.85 μL
Total	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Agarose gel electrophoresis of PCR fragments for constrcut J as well as C, P, Q, U & V

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, backbone PCR fragment for construct C (AR301-LC-57aaGS-HC) using primers 071 - 074, backbone PCR fragment for construct C using primers 072 - 073 & 075 - 076, backbone PCR fragment for construct P (cG250-LC), empty lane, backbone PCR fragment for construct Q (cG250-HC), backbone PCR fragment for construct U (006.11-LC), backbone PCR fragment for construct V (006.11-HC), empty lane, ladder, empty lane, backbone PCR fragment for construct J (scFvhu128.1-11aaAPG-scFvAR301-HC), empty lane, insert PCR fragment for construct J, empty lane, ladder, ladder. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the case of construct J. At the same time, only a low amount of desired product could be confirmed for every other sample.

DH5α transformation with C, P, Q, U, V & plating

Goal

Transformation was performed to introduce plasmid DNA constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

No or little to no colonies were present on the selective agar plate after overnight incubation.

Gibson assembly for C, P, Q, U & V

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	C	C’	P	Q	U	V
Master mix	5 μL	5 μL	5 μL	5 μL	5 μL	5 μL
Vector	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL	100 ng / 2 μL
Insert	107 ng / 2.15 μL	107 ng / 2.15 μL	27 ng / 1.35 μL	25 ng / 1.25 μL	27 ng / 1.35 μL	28.5 ng / 1.43 μL
ddH2O	0.85 μL	0.85 μL	1.65 μL	1.75 μL	1.65 μL	1.57 μL
Total	10 μL	10 μL	10 μL	10 μL	10 μL	10 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

DpnI digest for C, P, Q, U & V

Goal

Restriction enzyme digest with DpnI was performed to cleave methylated plasmid DNA of the PCR samples for constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC).

Procedure

Digest was carried out using the DpnI restriction enzyme (New England Biolabs) according to the manufacturer’s instructions. 1 μg of DNA was combined with the 5 μL of 10X rCutSmart Buffer (New England Biolabs) in a 1.5 mL reaction vessel. Following the addition of 20 units of the restriction enzyme, the reaction mix was incubated at 37 °C for 5 - 15 min. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Amount
DNA	1 μg
10X rCutSmart Buffer	5 μL
DpnI	1 μL
ddH2O	up to 50 μL
Total	50 μL

Finally, the enzyme was heat-inactivated at 80 °C for 20 min.

Results

The efficiency of the digest reaction was evaluated by subsequent experiments.

PCR purification for C, P, Q, U & V

Goal

A PCR clean-up of backbone and insert PCR fragments for constructs C (AR301-LC-2A-HC), P (cG250-LC), Q (cG250-HC), U (006.11-LC), and V (006.11-HC) was performed to purify DNA from the PCR reaction for downstream applications.

Procedure

PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) following modified manufacturer’s instructions. An equal volume of Membrane Binding Solution (Promega) was added to the PCR reaction mix, and the mixture was thoroughly mixed by vortexing. The mixture was then transferred to an SV Minicolumn placed in a collection tube, and incubated for 1 min at room temperature. The column was centrifuged at 16000 × g or 13053 rpm (Centrifuge 5425; Eppendorf) for 1 min to allow the DNA to bind to the column. The flow-through was discarded, and the column was washed with 700 μL and subsequently again with 500 μL Membrane Wash Solution (Promega) supplemented with 95% ethanol (4:1) at 16000 × g for 1 min each to remove impurities and contaminants. Excess ethanol was removed by centrifuging the column for additional 5 min at 16000 × g. Purified PCR products were eluted into a 1.5 mL microcentrifuge tube by adding 25 μL of nuclease-free water to the column matrix, incubating at room temperature for 3 min, and centrifuging the column for 1 min at 16000 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The clean-up procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Product	Concentration (ng/μL)

Sanger sequencing of construct I

Goal

DNA sequencing was performed to confirm the nucleotide sequence of construct I (scFvAR301-HC) using PCMV_fw and B4_R_AR301-VH_IgG1-CH primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06765737	048_PCMV_fw	130_I_scFvAR301-HC	Confirmed
06765738	064_B4_R_AR301-VH_IgG1-CH	130_I_scFvAR301-HC	Confirmed
06765739	048_PCMV_fw	130_I’_scFvAR301-HC	Confirmed
06765740	064_B4_R_AR301-VH_IgG1-CH	130_I’_scFvAR301-HC	Confirmed

Q5 PCR for construct J

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from 130_I_scFvAR301-HC and 009_scFv-hu128.1 using primers 081_D1_F_AR301-VL_11aaAPG, 141_J2_R_Leader(H)_hu128.1-VL and 142_J3_F_hu128.1-VL_Leader(H), 084_D4_R_hu128.1-VH_11aaAPG respectively for construct J (scFvhu128.1-11aaAPG-scFvAR301-HC) with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture is defined as follows:

Substance	130_I_scFvAR301	009_scFv-hu128.1
Master mix	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	10 ng / 1 μL
ddH2O	19 μL	19 μL
Total	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	5:00, 98 °C
Denaturation	1:00, 98 °C
Annealing	1:00, 68 - 70 °C
Extension	5:00, 72 °C
Final extension	10:00, 72 °C
Cycles	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Agarose gel electrophoresis of PCR fragments for constrcuts C, P, Q, U & V (Redo)

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, backbone and insert PCR fragments for construct C (AR301-LC-57aaGS-HC) using primers 071 - 074, backbone and insert PCR fragments for construct C using primers 072 - 073 & 075 - 076, insert PCR fragment for construct C, backbone and insert PCR fragments for construct P (cG250-LC), Ladder, backbone and insert PCR fragments for construct Q (cG250-HC), backbone and insert PCR fragments for construct U (006.11-LC), backbone and insert PCR fragments for construct V (006.11-HC). Due to the low loading volume, no new information could be derived from the gel.

Maxiprep of construct I.4 (Redo)

Goal

A maxiprep of construct I (scFvAR301-HC) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)
130_I_scFvAR301-HC	1	2750
130_I_scFvAR301-HC	2	2916

Agarose gel electrophoresis of PCR fragments for constrcuts C, P, Q, U & V

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, backbone and insert PCR fragments for construct C (AR301-LC-57aaGS-HC) using primers 071 - 074, 2 × backbone and insert PCR fragments for construct C using primers 072 - 073 & 075 - 076, insert PCR fragment for construct C, backbone and insert PCR fragments for construct P (cG250-LC), Ladder, backbone and insert PCR fragments for construct Q (cG250-HC), backbone and insert PCR fragments for construct U (006.11-LC), backbone and insert PCR fragments for construct V (006.11-HC). Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples, more so for inserts. Most backbone samples, however, have to be run through gel again.

Q5 PCR for constructs P, Q, U & V

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from 001_MOR4649-LC and 015_scFv-cG250 using primers 201_P1_F_IgL2-CL_cG250-VL, 202_P2_R_Leader(L)_cG250-VL and 203_P3_F_cG250-VL_Leader(L), 204_P4_R_cG250-VL_IgL2-CL respectively for construct P (cG250-LC), from 002_MOR4649-HC and 015_scFv-cG250 using primers 211_Q1_F_IgG1-CH_cG250-VH, 212_Q2_R_Leader(H)_cG250-VH and 213_Q3_F_cG250-VH_Leader(H), 214_Q4_R_cG250-VH_IgG1-CH respectively for construct Q (cG250-HC), as well as from 001_MOR4649-LC and 012_scFV-006-11 using primers 251_U1_F_IgL2-CL_006.11-VL, 252_U2_R_Leader(L)_006.11-VL and 253_U3_F_006.11-VL_Leader(L), 254_U4_R_006.11-VL_IgL2-CL respectively for construct U (006.11-LC), from 002_MOR4649-HC and 012_scFV-006-11 using primers 261_V1_F_IgG1-CH_006.11-VH, 262_V2_R_Leader(H)_006.11-VH and 263_V3_F_006.11-VH_Leader(H), 264_V4_R_006.11-VH_IgG1-CH respectively for construct V (006.11-HC) with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture for each reaction is defined as follows:

Substance	Backbone	Insert
Master mix	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	10 ng / 1 μL
ddH2O	19 μL	19 μL
Total	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	5:00, 98 °C
Denaturation	1:00, 98 °C
Annealing	1:00, 67 - 74 °C
Extension	5:00, 72 °C
Final extension	10:00, 72 °C
Cycles	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Q5 PCR for construct C (Redo)

Goal

Polymerase chain reaction using Q5 DNA polymerase was performed to amplify specific DNA fragments from 050_A_AR301-LC and 060_B_AR301-HC using primers 071_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A and 073_C3_F_Leader(H)_2A, 074_C4_R_IgG1-CH_pcDNA respectively as well as primers 075_C1_F_pcDNA_IgG1-CH, 072_C2_R_IgL2-CL_2A and 073_C3_F_Leader(H)_2A, 076_C4_R_IgG1-CH_pcDNA respectively for construct C (AR301-LC-2A-HC) with high fidelity and efficiency.

Procedure

PCR was carried out using the Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. For this, 1 - 1000 ng DNA template, 25 pmol forward and reverse primers, and 25 μL of the master mix were combined in a PCR tube. DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mixture for each reaction is defined as follows:

Substance	Backbone	Insert
Master mix	25 μL	25 μL
10 μM forward primer	2.5 μL	2.5 μL
10 μM reverse primer	2.5 μL	2.5 μL
Template DNA	100 ng / 1 μL	10 ng / 1 μL
ddH2O	19 μL	19 μL
Total	50 μL	50 μL

The reaction mixture was subjected to thermal cycling (T100 Thermal Cycler or C1000 Touch Thermal Cycler; Bio-Rad), which included 25 - 35 denaturation, annealing, and extension steps optimized in their duration for specific requirements of the DNA template. Optimal annealing temperature for the reaction was determined from the lower melting temperature of the primer pair used following the formula T_a(^°_C)=T_m(^°_C)+1~^°_C, or using the tool, NEB Tm Calculator (New England Biolabs). Resulting thermocycling conditions were:

Step	Condition
Initial denaturation	5:00, 98 °C
Denaturation	1:00, 98 °C
Annealing	1:00, 67 - 74 °C
Extension	5:00, 72 °C
Final extension	10:00, 72 °C
Cycles	×34

Amplified DNA products were kept at 4 - 12 °C until further use.

Results

The efficiency of the PCR was evaluated by subsequent experiments.

Maxiprep of construct I.4

Goal

A maxiprep of construct I (scFvAR301-HC) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

No DNA was obtained from the maxiprep, as the DNA failed to precipitate after centrifugation with isopropanol and subsequently with ethanol.

DH5α transformation with constract I.4 & suspension culture

Goal

Transformation was performed to introduce plasmid DNA construct I (scFvAR301-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells were transferred to 500 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the transformed cells.

Results

Successful transformation was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

Sanger sequencing of construct I.4 (Redo)

Goal

DNA sequencing was performed to confirm the nucleotide sequence of construct I (scFvAR301-HC) using PCMV_fw and B4_R_AR301-VH_IgG1-CH primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06765735	048_PCMV_fw	130_I_scFvAR301-HC / 4	Confirmed
06765736	064_B4_R_AR301-VH_IgG1-CH	130_I_scFvAR301-HC / 4	Confirmed

Sanger sequencing of construct C (Redo)

Goal

DNA sequencing was performed to confirm the nucleotide sequence of construct C (AR301-LC-TA-HC) using PCMV_fw and B4_R_AR301-VH_IgG1-CH primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis did not confirm a match between the experimental and expected sequences, although the sequencing data showed a high quality score and a continuous read length of over 1 kb. Due to the use of a different forward primer, which is binding further upstream of the insert, previously reported uncertainty within the light chain leader sequence was cleared. Sequencing of the T2A site with a different reverse primer, located closer downstream, still showed base pair deletion.

Barcode	Primer	Sample	Validation
06765733	048_PCMV_fw	070_C_AR301-LC-2A-HC	No uncertainty within light chain leader, base pair deletion within the T2A site
06765734	064_B4_R_AR301-VH_IgG1-CH	070_C_AR301-LC-2A-HC	No uncertainty within light chain leader, base pair deletion within the T2A site

Sanger sequencing of construct C (Redo)

Goal

DNA sequencing was performed to confirm the nucleotide sequence of construct C (AR301-LC-TA-HC) using NB_091_fw and NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis did not confirm a match between the experimental and expected sequences, although the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06765731	046_NB_091_fw	070_C_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site
06765732	047_NB_092_rev	070_C_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site

Sanger sequencing of constructs C, D, E & I (Redo)

Goal

DNA sequencing was performed to confirm the nucleotide sequences of constructs C (AR301-LC-2A-HC), D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC) using NB_091_fw and NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis confirmed a match between the experimental and expected sequences. In addition, the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06765695	046_NB_091_fw	070_C_AR301-LC-2A-HC / 1	Uncertainty within light chain leader and IgG1 constant region, base pair deletion within heavy chain leader
06765696	047_NB_092_rev	070_C_AR301-LC-2A-HC / 1	Uncertainty within light chain leader and IgG1 constant region, base pair deletion within heavy chain leader
06765697	046_NB_091_fw	070_C_AR301-LC-2A-HC / 2	Failed insertion, religation of construct A
06765698	047_NB_092_rev	070_C_AR301-LC-2A-HC / 2	Failed insertion, religation of construct A
06765699	046_NB_091_fw	070_C_AR301-LC-2A-HC / 3	Failed insertion, religation of construct A
06765700	047_NB_092_rev	070_C_AR301-LC-2A-HC / 3	Failed insertion, religation of construct A
06765701	046_NB_091_fw	070_C_AR301-LC-2A-HC / 4	Failed insertion, religation of construct A
06765702	047_NB_092_rev	070_C_AR301-LC-2A-HC / 4	Failed insertion, religation of construct A
06765703	046_NB_091_fw	070_C_AR301-LC-2A-HC / 5	Failed insertion, religation of construct A
06765704	047_NB_092_rev	070_C_AR301-LC-2A-HC / 5	Failed insertion, religation of construct A
06765705	046_NB_091_fw	070_C_AR301-LC-2A-HC / 6	Failed insertion, religation of construct B
06765706	047_NB_092_rev	070_C_AR301-LC-2A-HC / 6	Failed insertion, religation of construct B
06765707	046_NB_091_fw	070_C_AR301-LC-2A-HC / 7	Failed insertion, religation of construct A
06765708	047_NB_092_rev	070_C_AR301-LC-2A-HC / 7	Failed insertion, religation of construct A
06765719	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1	Failed insertion, religation of construct C
06765720	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1	Failed insertion, religation of construct C
06765721	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2	Failed insertion, religation of construct C
06765722	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2	Failed insertion, religation of construct C
06765723	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3	Failed insertion, religation of construct C
06765724	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3	Failed insertion, religation of construct C
06765725	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1	Uncertainty within light chain leader and insert
06765726	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1	Uncertainty within light chain leader and insert
06765727	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2	Failed insertion, religation of construct C
06765728	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2	Failed insertion, religation of construct C
06765729	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3	Failed insertion, religation of construct C
06765730	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3	Failed insertion, religation of construct C
06765709	046_NB_091_fw	130_I_scFvAR301-HC / 1	Uncertainty within light chain leader and IgG1 constant region, nonconservative point mutation (G174R) in AR-301 heavy chain variable region
06765710	047_NB_092_rev	130_I_scFvAR301-HC / 1	Uncertainty within light chain leader and IgG1 constant region, nonconservative point mutation (G174R) in AR-301 heavy chain variable region
06765711	046_NB_091_fw	130_I_scFvAR301-HC / 2	Failed insertion, religation of guselkumab backbone
06765712	047_NB_092_rev	130_I_scFvAR301-HC / 2	Failed insertion, religation of guselkumab backbone
06765713	046_NB_091_fw	130_I_scFvAR301-HC / 3	Failed insertion, religation of guselkumab backbone
06765714	047_NB_092_rev	130_I_scFvAR301-HC / 3	Failed insertion, religation of guselkumab backbone
06765715	046_NB_091_fw	130_I_scFvAR301-HC / 4	Uncertainty within light chain leader, AR-301 heavy chain variable region, and IgG1 constant region
06765716	047_NB_092_rev	130_I_scFvAR301-HC / 4	Uncertainty within light chain leader, AR-301 heavy chain variable region, and IgG1 constant region
06765717	046_NB_091_fw	130_I_scFvAR301-HC / 5	Failed insertion, religation of guselkumab backbone
06765718	047_NB_092_rev	130_I_scFvAR301-HC / 5	Failed insertion, religation of guselkumab backbone

Sanger sequencing of constructs C, D, E & I

Goal

DNA sequencing was performed to confirm the nucleotide sequences of constructs C (AR301-LC-2A-HC), D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC) using NB_091_fw and NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

No results were obtained from the sequencing service provider, most likely due to the loss of the samples.

Barcode	Primer	Sample	Validation
06764138	046_NB_091_fw	070_C_AR301-LC-2A-HC / 1
06764139	047_NB_092_rev	070_C_AR301-LC-2A-HC / 1
06764140	046_NB_091_fw	070_C_AR301-LC-2A-HC / 2
06764141	047_NB_092_rev	070_C_AR301-LC-2A-HC / 2
06764142	046_NB_091_fw	070_C_AR301-LC-2A-HC / 3
06764143	047_NB_092_rev	070_C_AR301-LC-2A-HC / 3
06764144	046_NB_091_fw	070_C_AR301-LC-2A-HC / 4
06764145	047_NB_092_rev	070_C_AR301-LC-2A-HC / 4
06764146	046_NB_091_fw	070_C_AR301-LC-2A-HC / 5
06764147	047_NB_092_rev	070_C_AR301-LC-2A-HC / 5
06764148	046_NB_091_fw	070_C_AR301-LC-2A-HC / 6
06764149	047_NB_092_rev	070_C_AR301-LC-2A-HC / 6
06765656	046_NB_091_fw	070_C_AR301-LC-2A-HC / 7
06765657	047_NB_092_rev	070_C_AR301-LC-2A-HC / 7
06765658	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1
06765659	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 1
06765660	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2
06765661	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 2
06765662	046_NB_091_fw	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3
06765663	047_NB_092_rev	080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC / 3
06765664	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1
06765665	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 1
06765666	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06765667	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 2
06765668	046_NB_091_fw	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3
06765669	047_NB_092_rev	090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC / 3
06765670	046_NB_091_fw	130_I_scFvAR301-HC / 1
06765671	047_NB_092_rev	130_I_scFvAR301-HC / 1
06765672	046_NB_091_fw	130_I_scFvAR301-HC / 2
06765673	047_NB_092_rev	130_I_scFvAR301-HC / 2
06765674	046_NB_091_fw	130_I_scFvAR301-HC / 3
06765675	047_NB_092_rev	130_I_scFvAR301-HC / 3
06765676	046_NB_091_fw	130_I_scFvAR301-HC / 4
06765677	047_NB_092_rev	130_I_scFvAR301-HC / 4
06765678	046_NB_091_fw	130_I_scFvAR301-HC / 5
06765679	047_NB_092_rev	130_I_scFvAR301-HC / 5

Miniprep of constructs C, D, E & I

Goal

A miniprep of constructs C (AR301-LC-2A-HC), D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC) was performed to isolate a small amount of plasmid DNA for downstream applications.

Procedure

DNA plasmids were prepared using the PureYield Plasmid Miniprep System (Promega) following modified manufacturer’s instructions. Briefly, 2 × 1.5 mL of overnight E. coli culture were centrifuged at 21300 × g or 15060 rpm (Centrifuge 5425; Eppendorf) for 30 s, and the resulting pellet was resuspended in 600 μL of water. Cell lysis was achieved by adding 100 μL of Cell Lysis Buffer (Promega), followed by 4 - 6 times inversions. The lysed cells were neutralized with 350 μL of pre-cooled Neutralization Solution (Promega) and centrifuged at 21300 × g for 3 min. The supernatant was applied to a PureYield-Minicolumn, which was then centrifuged at 21300 × g for 15 s to discard the flowthrough. The column was washed by adding 200 μL of Endotoxin Removal Wash (Promega) and centrifuging for 15 s at 21300 × g, followed by 400 μL of Column Wash Solution (Promega) supplemented with 95% ethanol (4:1) and centrifugation for 30 s at 21300 × g. Excess ethanol was removed by centrifuging the column for additional 5 min at 21300 × g. The DNA was eluted into a 1.5 mL microcentrifuge tube by adding 35 μL of nuclease-free water to the column matrix, incubating at room temperature for 1 min, and centrifuging the column for 15 s at 21300 × g. DNA concentration was determined using a NanoPhotometer N60 (Implen), and the DNA samples were stored at −20 °C.

Results

The miniprep procedure yielded DNA of sufficient quantity and purity ratios A260/A280 and A260/A230 within the acceptable range.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	1	120.1
070_C_AR301-LC-2A-HC	2	323.15
070_C_AR301-LC-2A-HC	3	331.00
070_C_AR301-LC-2A-HC	4	423
070_C_AR301-LC-2A-HC	5	281.1
070_C_AR301-LC-2A-HC	6	437.8
070_C_AR301-LC-2A-HC	7	300.25
080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC	1	408.00
080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC	2	295.45
080_D_scFvhu128.1-11aaAPG-AR301-LC-2A-HC	3	456.50
090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC	1	326.60
090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC	2	338.40
090_E_AR301-LC-11aaAPG-scFvhu128.1-2A-HC	3	207.10
130_I_scFvAR301-HC	1	434.85
130_I_scFvAR301-HC	2	749.85
130_I_scFvAR301-HC	3	252.70
130_I_scFvAR301-HC	4	649.00
130_I_scFvAR301-HC	5	241.90

DH5α colony picking constructs C, D, E & I

Goal

Colony picking of individual DH5α E. coli bacterial colonies transformed with constructs C (AR301-LC-2A-HC), D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC) from selection plate was to performed for the replication and expression of the desired genetic material.

Procedure

Using a sterile inoculation loop or pipette tip, individual colonies were carefully picked from the surface of the selection plate. Each colony was transferred to 4 mL of fresh culture medium (LB medium with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C with continuous shaking at 200 rpm (Innova 42 Incubator Shaker; New Brunswick Scientific) for further growth and amplification of the selected cells. The agar plate was sealed to prevent contamination.

Results

Successful colony picking was confirmed by the observed turbidity of the inoculated liquid bacterial culture.

DH5α transformation with constructs D, E, I & plating

Goal

Transformation was performed to introduce plasmid DNA constructs D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC) into DH5α E. coli cells, allowing for the replication and expression of the desired genetic material.

Procedure

An aliquot of competent DH5α E. coli cells (New England Biolabs) stored at −80 °C was thawed on ice. The desired plasmid DNA, at a concentration of 10 - 100 ng/μL, was added to the cells (1 - 5 μL) and gently mixed by flicking the tube 4 - 5 times. The cell-DNA mixture was incubated on ice for 10 min. Following the incubation period, transformed cells spread on selective plates (LB agar plates with 100 μg/mL ampicillin; Sigma) and incubated overnight at 37 °C (IN30 Incubator; Memmert) to allow for colony growth.

Results

Successful transformation was confirmed by the presence of colonies on the selective agar plate.

Gibson assembly of constructs D, E & I

Goal

Gibson assembly was performed to assemble the respective vector and insert DNA fragments into constructs D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), and I (scFvAR301-HC).

Procedure

Assembly reaction was carried out using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) according to the modified manufacturer’s instructions. DNA fragments of interest, including 50 - 100 ng of vector with 2 - 5-fold molar excess of insert(s), were combined with the 5 μL of master mix in a 1.5 mL reaction vessel. Molar amount of each fragment was calculated following the formula n~(pmols)=m~(ng)×_{1,000N~(bp)×650~Da/bp}, or using the tool, NEBiocalculator (New England Biolabs). DNA concentration was determined using a NanoPhotometer N60 (Implen). The final composition of the reaction mix is defined as follows:

Substance	Construct D	Construct E	Construct I
Master mix	10 μL	10 μL	10 μL
Vector	104.7 ng / 2.5 μL	109.8 ng / 3 μL	90.45 ng / 3 μL
Insert	47.55 ng / 1.5 μL	54.42 ng / 1.5 μL	51.3 ng / 1.5 μL
ddH2O	6 μL	5.5 μL	5.5 μL
Total	20 μL	20 μL	20 μL

The reaction mixture was then incubated at 50 °C for 60 min (ThermoMixer F1.5; Eppendorf), allowing for the efficient assembly of the DNA fragments into a single construct. Following the incubation, samples were stored on ice or at −20 °C for subsequent transformation.

Results

The efficiency of the assembly reaction was evaluated by subsequent experiments.

Agarose gel electrophoresis of PCR fragments for constrcuts D, E, F

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, empty lane, insert PCR fragment for construct F (AR301-LC-57aaGS-HC), backbone PCR fragment for construct F, insert PCR fragment for construct D (scFvhu128.1-11aaAPG-AR301-LC-2A-HC), backbone PCR fragment for construct D, insert PCR fragment for construct E (AR301-LC-11aaAPG-scFvhu128.1-2A-HC), backbone PCR fragment for construct E. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples. A byproduct can be detected in backbone PCR fragments of constructs F and D, it is, however, of unclear origin. No product is observed for insert PCR fragment of construct F. This is most likely due to the lack of synthesized product of the ordered sequence.

Agarose gel electrophoresis of PCR fragments for constrcut I

Goal

Agarose gel electrophoresis was performed to visualize DNA fragments of the selected samples based on their size.

Procedure

Agarose gel electrophoresis was performed using a 0.8% w/v agarose gel (SERVA) prepared in 1 × TAE (Tris-acetate-EDTA) buffer and supplemented with 10 μl of 1 × HiSens Stain G (SERVA) nucleic acid stain. The gel was cast in a gel electrophoresis chamber and allowed to solidify. DNA samples mixed with loading dye (1:5; Gel Loading Dye, Purple (6X), no SDS; New England Biolabs) were loaded into the wells of the gel. 5 - 10 μL of a DNA ladder (1 kb Plus DNA Ladder; New England Biolabs) was also included as a size reference. The gel was submerged in the electrophoresis chamber filled with 1 × TAE buffer and connected to a power supply. A constant voltage of 120 V was applied, and the DNA fragments were allowed to migrate through the gel for 40 min. After electrophoresis, the DNA bands were visualized using an automated imaging system (Gel Doc EZ System; Bio-Rad).

Results

The following samples were loaded into the gel from left to right: ladder, insert PCR fragment for construct I (cFvAR301-HC), backbone PCR fragment for construct I, empty lane, 2 × unrelated DNA sample. Overall, the agarose gel electrophoresis analysis confirmed the presence and size distribution of the expected DNA fragments in the samples.

Sanger sequencing of construct C

Goal

DNA sequencing was performed to confirm the nucleotide sequence of construct C (AR301-LC-TA-HC) using NB_091_fw and NB_092_rev primers.

Procedure

DNA was sequenced using a Sanger sequencing service (Premix Express; GENEWIZ or LightRun NXP; Eurofins Genomics) by preparing and sending a DNA-primer mixture following the sample submission guidelines. 5 μL of purified plasmid at a concentration of 30 - 100 ng/μL or of purified PCR fragment at a concentration of 10 - 50 ng/μL was added to a 1.5 mL flip cap reaction tube, along with 5 μL of 5 μM primer solution. The tube was labeled with a barcode and delivered to a drop-off point.

Results

To verify the identity of the sequence, the obtained sequencing trace was aligned with the expected theoretical sequence. The alignment analysis did not confirm a match between the experimental and expected sequences, although the sequencing data showed a high quality score and a continuous read length of over 1 kb.

Barcode	Primer	Sample	Validation
06764137	046_NB_091_fw	070_C_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site
06764136	047_NB_092_rev	070_C_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site
06764134	046_NB_091_fw	070_C’_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site
06764133	047_NB_092_rev	070_C’_AR301-LC-2A-HC	Uncertainty within light chain leader, base pair deletion within the T2A site

Maxiprep of construct C

Goal

A maxiprep of construct C (AR301-LC-TA-HC) was performed to obtain a sufficient quantity of pure plasmid DNA for subsequent experiments.

Procedure

DNA plasmids were prepared using the QIAGEN Plasmid Mini, Midi, and Maxi kit according to the modified manufacturer’s instructions (QIAGEN). Overnight culture of E. coli was centrifuged at 4122 × g or 4200 rpm (Heraeus Megafuge 40R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The resulting pellet was resuspended in 10 mL of P1 Buffer (QIAGEN) supplemented with RNase A solution (100 μg/mL; QIAGEN), RNase Cocktail enzyme mix (100 μg/mL; Thermo Fisher Scientific), and LyseBlue reagent (1:1000; QIAGEN). The suspension was transferred to a 50 mL centrifuge tube. To lyse the cells, 10 mL of P2 Buffer (QIAGEN) was added and mixed by inverting 4 - 6 times, followed by incubation at room temperature for 5 min. The lysed cells were neutralized by adding 10 mL of pre-cooled P3 Buffer (QIAGEN) and mixing by inverting 4 - 6 times, followed by incubation on ice for 20 min. Lysis and neutralization were confirmed using the LyseBlue reagent, where the solution turned blue upon adding P2 Buffer and reverted to colorless on addition of P3 buffer with thorough mixing. The mixture was centrifuged at 18000 × g or 12442 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. The supernatant was filtered using a cell strainer (Cole-Parmer) and applied to the QIAGEN-tip500 column pre-equilibrated with 10 mL of QBT Buffer (QIAGEN). The column was then washed by adding 2 × 30 mL of QC Buffer (QIAGEN), and DNA was eluted with 15 mL of QF Buffer (QIAGEN) into a 50 mL centrifuge tube. DNA precipitation was performed by adding 10.5 mL of room-temperature isopropanol and centrifuging at 20000 × g or 13115 rpm (Heraeus Multifuge X1R Centrifuge; Thermo Fisher Scientific) for 30 min at 4 °C. After careful decanting of the supernatant, the DNA pellet was washed with 5 mL of room-temperature 70% v/v ethanol and centrifuged at 20000 × g for 60 min. Finally, the supernatant was aspirated using a vacuum pump without disturbing the pellet, and the pellet was air-dried for 5 - 10 min. The DNA was then redissolved in 300 - 500 μL of TE buffer, the concentration was determined using a NanoPhotometer N60 (Implen), and the DNA was stored at −20 °C.

Results

The performed maxiprep resulted in high DNA yield and purity ratios A260/A280 and A260/A230 within the acceptable range, indicating successful isolation of the desired plasmid DNA.

Plasmid	Replicate	Concentration (ng/μL)
070_C_AR301-LC-2A-HC	1	2850
070_C_AR301-LC-2A-HC	2	3352