From the iGEM Distrubion Kit plate, K4 (AB_Pp-pAOX1,BBF10K_000181) was resuspended with 10 µl of water. It was then incubated for 5 minutes and transferred into a PCR tube labeled "Pichia Vector".
PCR for the Backbone of AB_Pp-pAOX1,BBF10K_000181
2 µl of the resuspended AB_Pp-pAOX1,BBF10K_000181 was transferred into a PCR tube. 6 µl of ddH2O was then added and mixed in. Four PCR reactions were set up as follows:
On the left thermocycler with the 1st Q5 MasterMix
On the left thermocycler with the 2nd Q5 MasterMix
On the right thermocycler with the 1st Q5 MasterMix
On the right thermocycler with the 2nd Q5 MasterMix
The PCR was carried out following the protocol and as described in the table.
Step
Temperature
Time
Initial Denaturation
98°C
30 s
Cycle Denaturation
98°C
10 s
Cycle Annealing
64°C
30 s
Cycle Elongation
72°C
105 s
GoTo Step 2
30 times
Final Extension
70°C
150 s
Hold
4°C
ever
Notes:
A big thank you to the Freiburg team for allowing us to borrow their plate, as our iGEM package had been delayed.
Date: Tuesday, 07.06.2023
Participants: Jana, Sarah V., Sumohit
Summary:
Due to issues with the autoclave and unavailability of TAE buffer, the planned work for the day had to be canceled and delayed.
Date: Wednesday, 08.06.2023
Participants: Jana, Jessica, Sumohit
Summary:Medium autoklav. Ampicillin solution preparation. Agar plates poured.
Medium autoklav
1x LB-Medium 500 ml
1x LB-Medium 250 ml
1x LB-Agar 500 ml
Autoclaved at IBVT
Ampicillin solution preparation
5 ml Ampicillin prepared (100 mg/ml)
Agar plates poured
Agar plates poured:
5x without Amp.
~13 / 14 plates with Amp. (not sure about the exact count)
Stored in refrigerator 1.366
Date: Tuesday, 13.06.2023
Participants: Jana, Sarah V., Sumohit
Summary:Agarose gel electrophoresis.
Agarose gel electrophoresis
0.8% gel for PCR check of Pichia vector from Distribution Kit
Use of Loading dye with SDS
10 μl of sample from the PCR tubes
See protocol
Gel loading:
1kb plus on right vector block
Vektor Block links
M
MM1
MM2
MM1
MM2
Must repeat gel (without pBad vector)
Transformation of iGEM Biobricks in E. coli Sure cells.
Heatshock Transformation in E. coli SURE cells
(Procedure as per protocol)
2 setups of Pichia vector (from iGEM Kit)
1 setup of Pichia vector dilution
2 setups of pBad vector (iGEM 2021)
plated on LB-Amp plates & incubated at 30°C
Date: Wednesday, 14.06.2023
Participants: Jana, Sarah S.
Summary:Investigation of the transformation.
Investigation of the transformation
Examined plates
Some colonies grew on pBAD 44.5
Only 2 colonies on pBAD 27.5
No colonies on all Pichia vectors
Plates further incubated
Date: Thursday, 15.06.2023
Participants: Jana, Anna
Summary:
Reviewed plates
No changes/status since Wednesday, June 14th
Medium
PCR of Pichia vector
Same progress as on June 5th
PCR product from June 5th used as a template
Agarose gel
0.8% gel for PCR check of Pichia vector from Distribution Kit
Used Loading dye with SDS
10 μl of sample from the PCR tubes
See protocol
Gel image & loading scheme:
1kb plus
Pichia Vector (MM1 2021)
Pichia Vector (MM1 2021)
Pichia Vector (MM2 2021)
Pichia Vector (MM2 2021)
M
PCR clean-up (NEB Kit)
Setups 3 and 4 (total 45 μl)
Followed the instructions in the kit (-> DNA under 2 kb, centrifugation at 13000 rpm)
Elution in 12 μl
Measure concentration on NanoDrop!
Inoculate MiniPrep (from the transformation on June 13)
5 ml LB-Amp medium
Pick 3 colonies (pBAD-vector 44.5 ng/ml)
Overnight incubation at 37°C in shaker
New heat-shock transformation
3 setups each in SURE cells and NEB cells
Heat shock for 45 sec (standard protocol)
Heat shock for 30 sec
with centrifugation after recovery (13000 rpm, 1 min, 4°C -> discard supernatant and resuspend in 100 μl outgrowth medium)
Plate on Amp plates (200 μl or all for centrifuged sample)
Incubation at 30°C
Plate NEB and SURE cells without transformation on Agar plates (without Amp)
Incubation at 30°C
Notes:
A big thank you to the Freiburg team for allowing us to borrow their plate, as our iGEM package had been delayed.
Date: Friday, 16.06.2023
Participants:: Anna, Sumohit
Summary:Plasmid isolation of the pBAD vector. Transformation with a new protocol. Sequencing.
Received Falcons and tip boxes + tips from Stutz, as well as large tip boxes from Biochemistry
Poured new agar plates with 100 μg/ml ampicillin
Plasmid isolation
Plasmid isolation from ÜN cultures of the pBAD vector (elution with 12 ul) and measurement of DNA concentration of the Pichia vector (2 setups) from PCR clean-up
Setup
Method
Concentration [ng/ul]
260/280
230/260
pBAD 1
PI
76.2
1.85
2.13
pBAD 2
PI
94.6
1.87
2.23
pBAD 3
PI
65.9
1.89
2.19
Pichia Vector PCR
PCR
108.0
1.91
2.39
Pichia Vector PCR
PCR
121.6
1.88
2.37
Transformation of 3 iGEM Pichia vectors
Transformation with a new protocol (see protocol) of 3 iGEM Pichia vectors
Plates left at room temperature over the weekend
Sequencing
Sequencing of Pichia vectors from PCR or PCR clean-up and pBAD vectors
Date: Monday, 19.06.2023
Participants: Jana, Sarah H.
Summary: Investigation of transformation. Plasmid isolation
Investigation of transformation
Colonies grown on all plates (3 Pichia vectors in NEB stable cells)
Plates in the refrigerator
Inoculation in 5 ml LB-medium + 100 μg/ml Amp (2 setups per vector)
Overnight incubation at 30°C in the shaker
Task1
[Description of what happened in task 1]
Task 2
[Description of what happened in task 2]
Date: Tuesday, 20.06.2023
Participants: Jana, Sarah V., Sumohit
Summary: Plasmid isolation. Sequencing of the isolated plasmids.
Plasmid isolation
Plasmid isolation (MiniPrep)
Conducted as per Kit Manual (see protocols)
Eluted in 50 μl
DNA concentration measured using NanoDrop
Sample
Concentration [ng/ul]
A260/280
A230/260
Pichia Vector 1.1
19.5
2.50
2.03
Pichia Vector 1.2
37.1
2.15
2.27
Pichia Vector 2.1
32.4
2.28
2.07
Pichia Vector 2.2
31.9
2.27
2.41
Pichia Vector 3.1
35.6
2.13
2.28
Pichia Vector 3.2
41.7
2.33
2.34
Pichia Vector 1: well K4
Pichia Vector 2: well E14
Pichia Vector 3: well G14
Sequencing
Sequencing
50-100 ng/μl DNA & 5 μM Primer
Since the concentration is so low:
7.5 μl Plasmid
2.5 μl Primer (10 μM)
Sample 1.2 forward (1): Seq. ID EMX401
Sample 1.2 reverse (2): Seq. ID EMX399
Sample 3.2 forward (1): Seq. ID EMX400
Sample 3.2 reverse (2): Seq. ID EMX398
Date: Wednesday, 21.06.2023
Participants: Jana, Sarah S.
Summary:
Overnight culture as on Monday, June 19th
Date: Thursday, 22.06.2023
Participants: Sarah S., Sumohit
Summary:Overnight culture
Overnight culture as on Monday, June 19th
Date: Friday, 23.06.2023
Participants: Sarah H., Sumohit
Summary: Restriction digestion of Minipreps with BSAI. Agarose gel electrophoresis. Plasmid isolation.
Restriction digestion of Minipreps with BSAI
Approach:
Component
Volume
ddH2O
ad 10 ul
BSAI
0.5 ul
CutSmart
1 ul
DNA (100 ng)
1.1: 5.13 ul 1.2: 2.7 ul 2.1: 3.09 ul 2.2: 3.13 ul 3.1: 2.8 ul 3.2: 2.4 ul
Incubation for 20 min at 37°C in PCR machine
Agarose gel electrophoresis
0.7% Agarose Gel according to the protocol
Scheme:
M M1.1 M1.2 M2.1 M2.2 M3.1 M3.2 M
Plasmid isolation (MiniPrep)
Conducted as per Kit Manual (see protocols)
Eluted in 50 μl
DNA concentration measured using NanoDrop
Sample
Concentration [ng/ul]
A260/280
A230/260
Pichia Vector 1.1
53.8
1.87
1.90
Pichia Vector 1.2
45.1
1.70
1.48
Pichia Vector 2.1
92.1
1.93
2.24
Pichia Vector 2.2
63.8
1.97
2.36
Pichia Vector 3.1
69.4
1.82
1.90
Pichia Vector 3.2
76.5
1.99
2.10
Pichia Vector 1: well K4
Pichia Vector 2: well E14
Pichia Vector 3: well G14
Sequencing
50-100 ng/μl DNA & 5 μM Primer
Since the concentration is so low:
5 μl Plasmid
5 μl Primer (5 μM)
Sample 1.1 forward (1): Seq. ID EMX368
Sample 1.1 reverse (2): Seq. ID EMX367
Sample 2.1 forward (1): Seq. ID EMX366
Sample 2.1 reverse (2): Seq. ID EMX371
Sample 3.1 forward (1): Seq. ID EMX370
Sample 3.1 reverse (2): Seq. ID EMX369
Date: Monday, 26.06.2023
Participants: Jana, Jessica, Sarah H.
Summary: Preparation for competent cells. Preparation for PCR.
Preparation for competent cells
Prepared RF1 and RF2 buffer for competent cells as per the protocol
Operated and calibrated the pH meter from the laboratory; calibration solutions available at IBVT
Coordination with IBVT regarding Falcon centrifuge, cold room, and liquid nitrogen
Items rearranged as needed
Protocol for competent cells was specified; for inquiries, contact Sarah H.
Preparation for competent cells
Primer and fragment for AMP gel were dissolved and frozen
Notes:
[Additional notes]
Date: Tuesday, 27.06.2023
Participants: Jana, Sarah V.
Summary: Gibson Assembly of AMP-0 (PCR and Agarose gel electrophoresis). Preparing for making component cells
PCR
PCR: Gibson-Overhang Fragment
Fragment: Pre-experiment BB-41
Primers: Frag-AMP-forw & Alternative Fragment (diluted 1:10 -> 10μM)
Reaction Setup:
1.25 μl forward Primer
1.25 μl reverse Primer
1 μl Template DNA
12.5 μl Q5 High-Fidelity 2X Master Mix
9 μl water
PCR Program: AMPOVERH
(PCR Machine 1 -> right side)
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
Elongation
72
18
Final Extension
72
120
Hold
4
for ever
Agarose gel electrophoresis
Agarose Gel (1%) at 100V for 1 hour
1: 5 μl Marker
2: 10 μl PCR sample
Preparing for making component cells
NEB stable -> Preculture
Pick 1 culture
in 4 ml LB-Medium + 1% Glucose (without Amp!)
overnight at 37°C in shaker
Date: Wednesday, 28.06.2023
Participants: Jana, Jessica, Sarah S.
Summary: Making of competent cells
Making of competent cells
Preparation of competent cells as per the protocol.
OD values for cultivation:
Time
OD 600 Culture 1
OD 600 Culture 2
0h 0 min
0.04
0.04
45 min
0.08
0.08
90 min
0.23
0.24
105 min
0.33
0.34
120 min
0.43
0.54
50 μl aliquots of the competent NEB Stable cells are stored in the -70 Freezer, Box 5, in the bottom left of the tower in unlabeled boxes. The freezer is located in the laboratory.
Date: Thursday, 29.06.2023
Participants: Jessica, Sarah H.
Summary: Verification of the competence with different concetration and methods.
Verification of the competence.
Verification of the competence of self-made NEB Stable cells
Transformation of pESP plasmid according to the protocol with various plating volumes or centrifugation
Plating volumes:
100 μl plating volume
200 μl plating volume
Centrifuge in 50-100 μl and plate
Different plasmid amounts:
5 ng → 5 μl of 1ng/ul dilution
1 ng → 1 μl of 1ng/ul dilution
500 pg → 0.5 μl of 1ng/ul dilution
Date: Friday, 30.06.2023
Participants: Jessica, Sarah H.
Summary: Maintance.
Maintance
Cleaning and autoclaving various items → Clearing items from the dishwasher and drying cabinet on Monday
Dissolving enzyme fragments
1000 ng -> + 100 µl -> 10 ng/µl in ddH2O
Vortex
1-hour incubation (shaker under the bench)
Primers not found for this purpose / not yet arrived, hence no PCR
Notes:
[Additional notes]
Date: Tuesday, 04.07.2023
Participants: Anna, Sarah S., Sarah V.
Summary: Competent cells. PCR for Gibson Assembly. Overnight cultures.
Competent cells
Competent cells (buffer + o/n cultures)
Preparation of buffers for competent cells (new protocol by Antonin)
CC Buffer: 200 ml
CaCl2 (50 mM, 5.5 g/l) -> 1.1 g (dissolve first, then add Glycerol)
10% Glycerol (20 ml -> (Density = 1.25 g/ml) 25 g
CaCl2 (50 mM, 5.5 g/l) -> 1.1g in 200 ml
Sterile filtration (using vacuum)
PCR for Gibson Assembly
Primers for enzymes have arrived (dissolved + frozen)
PCR: Overhang for Gibbson from His4, Dextranase Split1, and Split 2
Primer dissolution for enzymes (100 µM)
Vektor Gibson Primer for + rev
His4 for + rev
Mutanase for + rev
Dispersin B for + rev
Dextranase Split1 for + rev
Dextranase Split 2 for + rev
PCR: His4, Dextranase Split1, Dextranase Split2
His4 [2576 bp] -> His4over program (Device: PCR 1)
Overnight cultures for competent cells (NEB Stable)
Preculture from the cold room
Prepare 2x 10 ml LB-Medium (16:00)
Pick one colony each
Notes:
[Additional notes]
Date: Wednesday, 05.07.2023
Participants: Michael, Sarah S.
Summary: Making of competent cells. PCR
Making of competent cells
Competent cells were prepared according to Antonin's protocol. 200µl aliquots are stored at -80°C. A note is included in the bottom box of the rack.
PCR
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
8 s (30 cycles)
Annealing
70 °C
30 s (30 cycles)
Elongation
72 °C
80 s (30 cycles)
Final Extension
72 °C
2 min
Hold
4 °C
Date: Thursday, 06.07.2023
Participants:
Summary: PCR. Verification of competence. Gel electrophoresis of PCR products.
PCR:
25 µl setup
DNA template had no concentration specified - used 1 µl
Possibly used the wrong DNA template
Primer at 10 µM each - used 1.25 µl
2x Q5 High Fidelity Mix 12.5 µl
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
8 s (30 cycles)
Annealing
67 °C
30 s (30 cycles)
Elongation
72 °C
95 s (30 cycles)
Final Extension
72 °C
2 min
Hold
4 °C
Verification of competence of self-made NEB Stable Cells
Transformation of PESP plasmid following protocol (used 6 Eppis) with various plating volumes or centrifugation
- 3 Eppies centrifuged, discarded supernatant leaving 50 µl, resuspended in 300 µl, and plated with various volumes on different plates:
- Plating volume 200 µl
- Plating volume 100 µl
- 3 Eppies centrifuged, discarded supernatant leaving 50 µl, no new medium added, resuspended, and plated
Different plasmid amounts:
- 5 ng --> 5 µl of 1 ng/µl dilution
- 1 ng --> 1 µl of 1 ng/µl dilution
- 500 pg --> 0.5 µl of 1 ng/µl dilution
Gel electrophoresis of PCR products:
- Gel loaded with 10 µl sample and 2 µl loading dye
- Gel loading:
Ladder - His 4 - Mut - Dis B - Dex 1 - Dex 2 - Vec
Date: Friday, 07.07.2023
Participants: Michael, Sumohit
Summary: PCR.
PCR for Dispersin B, His4, and Mutanase with the recommended temperatures from NEB and SnapGene.
PCR Setup:
50 µl reaction volume
1 µl DNA template
Primer MasterMix with Rev and For Primer 5 µl
2x Q5 High Fidelity mix 25 µl
ddH2O 19 µl
Dispersin B and His4 (NEB)
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
10 s (30 cycles)
Annealing
69 °C
30 s (30 cycles)
Elongation
72 °C
85 s (30 cycles)
Final Extension
72 °C
3 min
Hold
4 °C
Dispersin B and His4 (SnapGene)
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
10 s (30 cycles)
Annealing
72 °C
30 s (30 cycles)
Elongation
72 °C
85 s (30 cycles)
Final Extension
72 °C
3 min
Hold
4 °C
Mutanase (NEB)
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
10 s (30 cycles)
Annealing
70 °C
30 s (30 cycles)
Elongation
72 °C
90 s (30 cycles)
Final Extension
72 °C
3 min
Hold
4 °C
Mutanase (SnapGene)
Action
Temperature
Time
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
10 s (30 cycles)
Annealing
70 °C
30 s (30 cycles)
Elongation
72 °C
90 s (30 cycles)
Final Extension
72 °C
3 min
Hold
4 °C
PCR clean-up of Pichia Vector:
Starting volume: 10 µl
Nanodrop measurement: -1.5 µl
Date: Monday, 10.07.2023
Participant:
Summary: PCR for Gibson Assembly
PCR
Dextranase Split 1:
2 setups:
2 different PCR programs
Concentrations in setups identical (see PCR protocol) - used fragment concentration: 10 ng/µl
Dex 1 [2166 bp] -> Program Dex1a (Device: PCR 1)
Step
Action
Temperature
Time
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s (30 cycles)
3
Annealing
70 °C
25 s
4
Elongation
72 °C
80 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Dex 1 [2166bp] -> DEX1BIGEM (Device: PCR in IBVT)
Step
Action
Temperature
Time
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s (30 cycles)
3
Annealing
68 °C
25 s
4
Elongation
72 °C
80 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Dextranase Split 2:
2 setups:
Dex 2 [1808bp] -> Program His4over (Device: PCR 1)
Step
Action
Temperature
Time
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s (30 cycles)
3
Annealing
72 °C
30 s
4
Elongation
72 °C
80 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Dex 2 [1808bp] -> Program His4over (Device: PCR 1)
Step
Action
Temperature
Time
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s (30 cycles)
3
Annealing
72 °C
25 s
4
Elongation
72 °C
80 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Notes:
[Additional notes]
Date: Tuesday, 11.07.2023
Participants: Sumohit, Wintana
Summary: Maintance.
Laboratory cleaned up
Notes:
[Additional notes]
Date: Wednesday, 12.07.2023
Date: Thursday, 13.07.2023
Participants: Sarah S., Sarah V., Sumohit
Summary: PCR.
PCR for pichia vector and His4.
PCR: Pichia Vector (1 ng / 3 ng / 5 ng)
[Description of the PCR with Pichia Vector at different concentrations]
PCR: HIS4
Fragment concentration: 5 ng/µl
Annealing at 67°C for 30 seconds, otherwise similar conditions
Notes:
[Additional notes]
Date: Friday, 14.07.2023
Participants: Sarah S., Sarah V., Sumohit
Summary: pH Verification of TAE Buffers. PCR. Agarose gel electrophoresis. PCR Gel Clean Up.
pH Verification of TAE Buffers
All three bottles are at ~8.25 pH.
It should ideally be between 8.3 and 8.5 (Antonin suggests we can still use them).
PCR: Dispersin Overhang
Fragment concentration: 5 ng/µl
Annealing at 67°C for 30 seconds, otherwise similar conditions
Agarose Gel 0.8%:
Samples include 3x Pichia Vector (from Thu, July 13), + His4 (from Thu, July 13) + Dispersin (from today, July 14).
Large gel chamber: 80 V for 1 h 45 min --> we made a mistake and then ran it again at 120 V for 30 min (next time, run at 120 V for ~1 h).
GelRed: Add 2 µl to 50 ml Gel, shortly before pouring.
Primer Test for Mutanase, Dispersin B, and Dextranase 1.
Primer Test for Mutanase, Dispersin B, and Dextranase 1 using BB41 Vorexp AMP Fragment. AMP Rev was used as the reverse primer for Dextranase. BB41 was used as a positive control.
Alternative PCR for Dispersin B using Mutanase's Primer and PCR Program:
Reaction Setup:
1.25 μl forward Primer
1.25 μl reverse Primer
1 μl Template DNA (AMP 10 ng, rest 3 ng)
12.5 μl Q5 High-Fidelity 2X Master Mix
9 μl Water
PCR Program: AMPOVERH (PCR Machine: iGEM)
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
30
Elongation
72
18
30
Final Extension
72
120
Hold
4
forever
PCR Program for Mutanase:
PCR Program: Mutanase68 (PCR Machine: IBVT)
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
forever
Sample Designations and Purposes:
2107-1: Mutanase Primer Test
2107-2: Dispersin B Primer Test
2107-3: Dextranase 1 Primer Test
2107-4: Mutanase Normal Repeat (Positive Control)
2107-5: Dispersin B Fragment Test
2107-6: BB41 AMP Normal Repeat (Positive Control)
Agarose-Gel 0.8%:
All samples from July 21
Small gel chamber: 80 V for 1 h
MidoriGreen: 2.5 µl in 50 ml Gel
1 µl Loading Dye added to 5 µl sample and then applied to the gel
Gel Order: Marker - Mutanase Primer Test - Dispersin B Primer Test - Dextranase 1 Primer Test - Mutanase - Dispersin B Fragment Test - BB41 AMP - Marker
Date: Monday, 24.07.2023
Participants:
Date: Tuesday, 25.07.2023
Participants: Anna, Sarah S.
Summary: Perform PCR and gel electrophoresis.
Perform PCR and gel electrophoresis
Preparation:
Mutanase H2O Template PCR
Reaction Mix (25µl):
1µl Template -> ddH2O
1.25µl of both Primers
12.5µl Mastermix
9µl ddH2O
His4 PCR
Reaction Mix (50µl):
2µl DNA (3ng/µl) -> total 6ng
2.5µl of both Primers
25µl Mastermix
18µl ddH2O
PCR Program:
Initial Denaturation: 98°C for 30s
Denaturation: 98°C for 8s
Annealing: 68°C, 67°C for 30s
Elongation: 72°C for 80s
Final Elongation: 72°C for 2min
Hold: 4°C
Dex2 PCR
Reaction Mix (50µl):
2µl DNA (3ng/µl) -> total 6ng
2.5µl of both Primers
25µl Mastermix
18µl ddH2O
Preparation:
Dye was added to the DNA tubes. 100 μL for Mutanase and 200 μL for His-4 and Dex-2. These were shaken to mix the dye better on the walls.
Order in Gel Electrophoresis:
Ladder
Mutanase
His-4
Dex-2
Notes:
U.V. table was used from the top, then cleaned and returned. DNA placed back in the top refrigerator.
Gel Result:
Date: Wednesday, 26.07.2023
Participants: Jana, Michael, Ronja
Goals: PCR. Agarose Gel Electrophoresis
Mut PCR - Gel Extraction - Determine Concentration
DispB PCR - Gel Extraction - Determine Concentration
Dex2 PCR Clean-up - Determine Concentration
Preparation:
Mut PCR
Reaction Mix (2x 50µl):
0.5µl DNA (10ng/µl) -> total 5ng
2.5µl of both Primers
25µl Mastermix
19.5µl ddH2O
PCR Program (Muto):
Initial Denaturation: 98°C for 30s
Denaturation: 98°C for 8s, 30 cycles
Annealing: 68°C for 30s, 30 cycles
Elongation: 72°C for 90s, 30 cycles
Final Extension: 72°C for 2min
Hold: 4°C
DispB PCR
Reaction Mix (2x 50µl):
0.5µl DNA (10ng/µl) -> total 5ng
2.5µl of both Primers
25µl Mastermix
19.5µl ddH2O
PCR Program:
Initial Denaturation: 98°C for 30s
Denaturation: 98°C for 8s, 30 cycles
Annealing: 68°C for 30s, 30 cycles
Elongation: 72°C for 80s, 30 cycles
Final Extension: 72°C for 2min
Hold: 4°C
Agarose Gel Electrophoresis
Gel Image:
Gel Extraction of Mutanase (new Zymo Kit)
Eluted in 10μl Elution Buffer
NanoDrop Concentration
Concentration [ng/ul] A260/280 A260/230
16.7 2.20 1.05
PCR Clean-up Dex2 (PCR from 25.07)
Eluted in 10μl
NanoDrop Concentration
Concentration [ng/ul] A260/280 A260/230
77.7 1.86 3.04
Date: Thursday, 27.07.2023
Participants: Jana, Jessica
PCR His4
Reaction Mix (2x 50µl):
2 µl DNA (3ng/µl) -> total 6 ng
2.5 µl rev Primer
2.5 µl for Primer
25 µl Q5 Mastermix
18 µl ddH2O
PCR Program:
Initial Denaturation: 98°C for 30s
Denaturation: 98°C for 8s, 30 cycles
Annealing: 67°C for 30s, 30 cycles
Elongation: 72°C for 80s, 30 cycles
Final Extension: 72°C for 2min
Hold: 4°C
PCR Dispersin B
Reaction Mix (2x 50µl):
1 µl DNA (3ng/µl) -> total 6 ng
2.5 µl rev Primer (Mutanase)
2.5 µl for Primer (Mutanase)
25 µl Q5 Mastermix
19 µl ddH2O
PCR Program (Mutanase68):
Initial Denaturation: 98°C for 30s
Denaturation: 98°C for 8s, 30 cycles
Annealing: 68°C for 30s, 30 cycles
Elongation: 72°C for 90s, 30 cycles
Final Extension: 72°C for 2min
Hold: 4°C forever
Agarose Gel Electrophoresis
Gel Image:
Gel Extraction Dispersin B
Method: Zymo Kit, eluted in 10 µl Elution Buffer (preheated to 50°C)
NanoDrop Concentration
Concentration [ng/ul] A260/280 A260/230
9.3 1.83 0.24
Amplification Pichia Vector
Reaction Mix (25 µl each for Pichia-Vector E14 and G14):
Experiment Title: PCR for His4 and Dextranase 1; Freezer Cleanup
Summary:
Various approaches were attempted for His4 PCR which did not yield successful results. The same holds true for Dextranase 1. Many items were removed and disposed of from the freezer during the cleanup.
Materials and Methods:
PCR Protocol Agarose Gel Electrophoresis Protocol PCR Purification Protocol Components:
Q5 2x Master Mix: 12.5 µl
10 µM Primer fw: 1.25 µl (0.5 µM)
10 µM Primer rv: 1.25 µl (0.5 µM)
ddH2O: 9 µl
DNA Template: 1 µl (10 ng DNA)
Primers for Mutanase, His4, Dextranase Split 1 Agarose Gel:
Agarose
1X TAE Buffer
Gel Chamber
Midori Green
PCR Variants:
His4 with His4 Primer:
Sample
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
His4 [2576 bp] -> His4 Program
Initial Denaturation
98
30
His4 [2576 bp] -> His4 Program
Denaturation
98
8
30
His4 [2576 bp] -> His4 Program
Annealing
69
20
30
His4 [2576 bp] -> His4 Program
Elongation
72
80
30
His4 [2576 bp] -> His4 Program
Final Extension
72
120
His4 [2576 bp] -> His4 Program
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
67
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
>
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
69
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
70
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Dextranase Split 1 with Mutanase For Primer and Dextranase 1 Rev Primer / Dextranase 1 Primer:
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
69
20
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
67
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
69
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Program Description
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
70
30
30
Elongation
72
80
30
Final Extension
72
120
Hold
4
for ever
Agarose Gel Electrophoresis with Pichia PCR Samples from 27th July
Gel Image:
Results:
Preparation:
A 0.8% agarose gel was used for agarose gel electrophoresis and run at 80 V for 60 minutes.
Gel Electrophoresis Order:
Ladder - Pichia Vector 1 - Pichia Vector 2 - His-4 - Dex-1 - Marker
...
Discussion:
The PCR for the Pichia vector was successful, showing clear bands in the gel and appropriate concentrations. In the His4 69°C 20 sec approach, a faint band is visible at the expected height, though uncertain. Hence, various annealing temperatures (67-70°C) were tested for 30 sec but were not applied to the gel due to time constraints. The wrong program was used (69°C 20 sec) as the previous entries were a bit cryptic, and the successful PCR was not clearly identified. For Dextranase 1 with Mutanase For Primer and Dextranase 1 Split Rec Primer, multiple bands are visible. The wrong size was read from a document (2367 instead of 2167 bp), and thus everything was merged with His4. Also, a gradient from 67°C to 70°C was created with two different primer combinations (Mut For-Dex1 Rev and Dex1 For-Dex1 Rev). The error was later identified.
For the Next Days:
- Repeat Dextranase 1 approaches with the correct elongation time of 60 sec.
- Apply all samples to the gel and analyze.
Date: Monday, 31.07.2023
Participants:: Sharkey and Sarah H.
Summary: Medium preparation. Dextranase 1 Gradient PCR. Gel Electrophoresis.
Medium preparation
Preparation of 500 ml YPD+His Medium and 200 ml YPD+His Agar using the following recipe:
Plates poured with YPD+His Agar and stored in the refrigerator at -U2.
YPD+His Medium stored in the laboratory.
Also, plates with yeast can be stored with bacteria in the refrigerator.
Dextranase 1 Gradient PCR
Gradient PCR from 62°C to 72°C using Mutanase forward primer and Dextranase1 reverse primer:
Summary: PCR. Pichia Pastoris Strain GS115. Gel Electrophoresis.
Performed His4 PCR with annealing at 67°C and 35 cycles.
Prepared fresh template and primer dilution for His4 PCR.
His4 PCR
Component
Volume (ul)
Q5 2x Master Mix
25
Forward Primer
2.5
Reverse Primer
2.5
Template
1
ddH2O
19
Program for His4 PCR:
Step
Action
Temperature (°C)
Duration
Cycles
1
Initial Denaturation
98
30 s
1
2
Denaturation
98
8 s
35
3
Annealing
67
30 s
35
4
Elongation
72
80 s
35
5
Final Extension
72
2 min
1
6
Hold
4
Dex1 PCR
Performed Dex1 PCR with annealing at 68-72°C, 30 cycles, and Mutanase forward primer.
Used fresh Mut Primer, fresh Dex1 reverse primer, and fresh Template Sample.
Component
Volume (ul)
Q5 2x Master Mix
25
Forward Primer
2.5
Reverse Primer
2.5
Template
1
ddH2O
19
Program for Dex1 PCR:
Step
Action
Temperature (°C)
Duration
Cycles
1
Initial Denaturation
98
30 s
1
2
Denaturation
98
8 s
30
3
Annealing
68-72°C in 1°C increments
30 s
30
4
Elongation
72
65 s
30
5
Final Extension
72
2 min
1
6
Hold
4
Pichia Pastoris Strain GS115
Spread Pichia Pastoris Strain GS115 on YPD+His plates with 3-streak and "Christmas tree" streaking, incubated at 30°C in the MiBi laboratory.
Christmas tree streaking
3-streak
Gel Electrophoresis
Performed gel electrophoresis on remaining Dex1 PCR samples from Monday and Friday, as well as the Dex1 fragment sample, to check for purity.
Notes:
Date: Wednesday, 02.08.2023
Participants: Jana, Jessie
Summary:
Performed agarose gel electrophoresis for His4 and Dex1 samples from August 1st.
Loaded 10 μl of His4 and 50 μl of Dex1 (68-72°C) into the gel, divided into 2 wells.
Task1: Gel Extraction for Dex1
Eluted Dex1 in 10 μl of elution buffer.
Task2: Concentration Analysis
Concentration [ng/ul]
A260/280
A260/230
114.0
1.90
2.08
Task3: Pichia Pastoris Overnight Culture
Prepared 4x 10 ml cultures in YPD + His Medium for Pichia Pastoris.
Inoculated at 4:30 PM.
Incubated overnight at 30°C on a shaker.
Note: Plates are stored in the IBVT refrigerator.
Notes:
Date: Thursday, 03.08.2023
Participants: Sumohit 09:30-18:00; Jana 09:30-18:00;
Summary:
Experiment: Gel for Münster; His4 PCR Variants; Competent Pichia Cells; Dex 2 PCR and Gel Electrophoresis to test the gel extraction kit.
Gel Preparation for Münster
Loaded samples of His4 and Dex1 (68-72°C) onto the agarose gel.
Gel Extraction for Dex1
Eluted Dex1 using 10 μl of elution buffer.
Concentration Analysis
Concentration [ng/ul] A260/280 A260/230
114.0 1.90 2.08
Pichia Pastoris Overnight Culture
Prepared 4x 10 ml cultures in YPD + His Medium for Pichia Pastoris.
Inoculated at 4:30 PM.
Incubated overnight at 30°C on a shaker.
Note: Plates are stored in the IBVT refrigerator.
His4 Variants
His4 samples with GC Enhancer 2 µl; His4 samples with 1µl 100% DMSO; His4 with both AMP BB41 Primers; His4 with BB41 Rev and His4 For; AMP BB41 with His4 Primer;
PCR Program for His4:
Step
Action
Temperature (°C)
Duration
Cycles
1
Initial Denaturation
98
30 s
1
Loading Scheme
Marker – pBAD18 – BB41 – BB41 with His4 Primer – Marker – Pichia Vector – Mutanase – Dispersin B – Dex1 excess from previous days – Dex1 Jana Gel Extraction – His4 100 ng from stock – Marker – His4 GC – His4 DMSO End Concentration 4% – His4 with BB41 Primer – His4 with BB41 Rev and His4 For – Marker – Dex2 – Dex2 – Dex2 – Dex2 – Marker
Competent Pichia Pastoris (Jana)
Allowed overnight cultures to grow at OD600 of 0.9 (at 30°C on shaker).
Prepared competent cells following the protocol in the Frozen Yeast Transformation Kit by Zymo.
Aliquoted competent cells in 200 μl aliquots. Cells can be refrozen after thawing (according to the kit) if the complete volume is not needed.
Stored in the -70°C freezer.
Notes:
Date: Friday, 04.08.2023
Date: Monday, 07.08.2023
Participants: Sarah 09:30-18:00; Sumohit 09:30-18:00;
Summary:Experiment: His4 PCR with different DMSO concentrations and adjusted annealing temperature and AMP; PCR for AMP Gibson Assembly; Testing old gel extraction kits and comparing with the new Zymo kit; Attempt to lower the A230 value using DNA Clean-Up and Concentrator Kit.
pBAD18 PCR
pBAD18
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40 Zyklen
Annealing
70
30
40 Zyklen
Elongation
72
180
40 Zyklen
Final Extension
72
3:30 min
Hold
4
AMP BB41 Pre-Experiment PCR
Alle BB41 (AMPOVERH)
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
30
Elongation
72
18
30
Final Extension
72
120
Hold
4
for ever
Task3: His4 PCR
Alle His4
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30 Zyklen
Annealing
67
30
30 Zyklen
Elongation
72
80
30 Zyklen
Final Extension
72
120
Hold
4
DMSO Concentration - Annealing Temperature [°C]
DMSO Concentration
Annealing Temperature [°C]
10%
61-63
Agarose Gel Preparation
Prepared a 0.6% agarose gel for His4 PCRs with peptide primers and loaded 25 ul in each well.
Prepared a 1.2% agarose gel for His4 DMSO PCRs, pBad, and AMP PCRs. Loaded 10 ul of His4 DMSO PCRs and 5 ul of pBad and AMP PCRs.
Testing Old and New Gel Extraction Kits
Used the old NEB Kit for extraction from 3 samples and the Zymo kit for 2 samples from the samples of Thursday, 3rd August.
Results
DNA Concentration and Ratios
Sample
DNA Concentration [ng/µl]
A260/A280
A260/A230
Kit
His4-Sample3
33,0
2,02
1,86
Alt NEB
Dex2-Sample1
33,2
1,88
0,57
Alt NEB
Dex2-Sample2
23,7
2,00
1,70
Zymo
Dex2-Sample3
15,0
2,01
1,03
Alt NEB
Dex2-Sample4
14,2
1,89
0,21
Zymo
Task6: DNA Clean-Up
Used DNA Clean-Up and Concentrator Kit to improve A260/A230 ratios for selected samples.
Results
DNA Concentration and Ratios
Sample
DNA Concentration [ng/µl]
A260/A280
A260/A230
Dex2-Sample1
7,9
2,03
3,11
Dex2-Sample2
3,9
1,80
11,05
Dex2-Sample3
-7,1
1,44
0,70
Discussion:
Both the old NEB Kit and the Zymo Kit worked well for one sample but showed poor A260/A230 ratios for another. Concentrations were above 15 ng/µl for all samples, and the A260/A280 ratios looked good. Considering possible organic contamination in some samples, DNA clean-up was performed. However, only Dex2-Sample1 showed good values after clean-up; other samples were unusable and were discarded. In the future, either the old NEB Kit or the Zymo Kit could be used for DNA extraction from gels.
Upcoming Tasks
Continue testing His4
Gibson Assembly for AMP BB41 in pBAD18
Date: Tuesday, 08.08.2023
Participants: Sumo, Jana, Sarah H.
Summary:Maintenance activities were conducted, including washing and autoclaving equipment. Additionally, PCR clean-up was performed for pBAD and AMP fragments with elution in 10 ul. Concentrations and ratios for the samples were measured.
DNA Concentration and Ratios
Sample
Concentration [ng/ul]
260/280
260/230
AMP
57,3
1,84
2,01
pBAD1
96,6
1,84
1,68
pBAD2
94,1
1,90
2,24
Gibson Assembly with NEBuilder for AMP BB41 in pBAD18
Components
Volume [ul]
Concentration (ng)
pBAD18
x (75 ng)
BB41
18.77 ng (OKS)
Water
Up to 10 ul
NEBbuilder
10 ul
Transformation
Transformation was carried out as per the protocol using 3µl and 5µl Gibson product from Lila Eppi, and 3µl Gibson product with NEBuilder, along with 1 µl pBAD18 as a control.
PCR with His4 Gel Extraction Product
Components
Volume [ul]
Q5 2xMM
25
fwd Primer
2.5
rev Primer
2.5
His4 Gel Extraction Template (100pg Verd.)
1/0.5/1 (1 ng/500 pg/100 pg)
ddH2O
19/19.5/19
His4 Gel Product (33.33 ng/ul) was diluted 1:33 in 33.0 ul to achieve a concentration of approximately 1 ng/ul. An additional 1:10 dilution was made from this dilution to obtain a concentration of 100 pg/ul.
PCR Program for His4
PCR Program for His4 Amplification
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
35
Annealing
67
30
35
Elongation
72
80
35
Final Extension
72
120
Hold
4
Date: Wednesday, 09.08.2023
Participants: Jana 09:30-17:00; Sumohit 09:30-17:00;
Summary: The Gibson Assembly plates were examined. Due to a high number of colonies on all three plates, 8 colonies from each plate were used for Colony-PCR and plated on a separate plate. All enzymes for BioBrick Assembly were checked for functionality. PCR was conducted with AMP primers for all fragments.
Plating after Gibson Assembly of BB41 into pBAD18
During Colony-PCR, 8 colonies from each plate were taken and checked using the AMP PCR program to confirm the presence of the desired insert in the cells.
Colony-PCR Program
PCR Program for iGEM PCR Machine
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
30
Elongation
72
18
30
Final Extension
72
120
Hold
4
forever
Mutanase, Dispersin B, Dextranase 1 and 2 PCR with AMP BB41 Primers
This was done to enable cloning into pBAD18 for BioBrick Assembly. For Dextranase 1, Dex1 Rev primer was used instead of AMP BB41 Rev, and for Dex2, Dex2 For primer was used instead of AMP BB41 For.
PCR Program for Mutanase and Dispersin B (PCR-Maschine IBVT)
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
forever
PCR Program for Dex2
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98 °C
30 s
Denaturation
98 °C
8 s
30 Zyklen
Annealing
72 °C
30 s
30 Zyklen
Elongation
72 °C
90 s
30 Zyklen
Final Extension
72 °C
2 min
Hold
4 °C
Preparation of 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, and Dex2 with AMP Primers
As the Colony PCR did not show any bands, overnight cultures were set with additional ampicillin to ensure its presence in case it was depleted from the plates.
Functionality Test of All Required Restriction Enzymes for BioBrick Assembly (except SpeI)
pBAD18 has restriction sites for EcoRI, XbaI as unique cutters, and PstI as dual cutters. To observe bands on the gel with EcoRI or XbaI digestion, an additional approach was made using BsaI, resulting in two bands on the gel. 500 ng of the sample was used for digestion.
Theoretical Expectation:
Order scheme: Marker- undigested (supercoii) - digest with EcoRi - digest with XbaI - digest with PstI - digest with EcoRI and BsaI - digest with XbaI and BsaI - marker
Preparation of 1% Agarose Gel for Functionality Test of All Required Restriction Enzymes for BioBrick Assembly (except SpeI and Dex1)
10 µl was applied.
Application Scheme:
Application scheme: Marker- undigested (supercoii) - Digest with EcoRi - Digest with XbaI - Digest with PstI - Digest with EcoRI and BsaI - Digest with XbaI and BsaI - Dex1 with AMP BB41 For and Dex1 Rev Primer
Discussion:
There were a high number of cells on the plates after Gibson Assembly, far exceeding the expected count. As a result, 8 colonies were selected from each plate for Colony-PCR to check if the insert was successfully cloned into the plasmid. The Colony-PCR and the corresponding gel confirmed that the Gibson Assembly was not successful. To validate the Colony-PCR results, overnight cultures were initiated and incubated. The functionality test of the restriction enzymes was successful and produced the expected results. The PCR with AMP BB41-pBAD18 overhangs worked for Dispersin B, Dex1 and 2, but not for Mutanase, and the reason is unknown. Considering potential issues with Gibson Assembly, preparations were made for BioBrick Assembly.
Next Steps:
Plasmid Isolation from the 24 overnight cultures and Restriction Analysis
Mutanase PCR
Preparation for BioBrick Assembly
Date: Thursday, 10.08.2023
Experiment: LB Agar Medium and LB Medium with Ampicillin and Chloramphenicol
Performed plasmid isolation, restriction analysis, and Gibson Assembly without transformation for subsequent BioBrick Assembly. PCR for BioBrick Assembly.
Summary: Suspecting that Ampicillin was not depleted, new plates and fresh Ampicillin medium were prepared. Plasmid isolation was performed on the 30 overnight cultures from the previous day, followed by restriction analysis. Gibson Assembly without transformation was conducted with all enzyme fragments into pBAD18. PCR was performed with AMP primers for Mutanase.
Plasmid Isolation Concentrations
Sample Concentrations and Ratios
Sample
Concentration
A260/A280
A260/A230
S1
36.31
1.93
2.22
S2
45.17
1.99
2.10
S3
36.5
1.88
2.05
S4
26.78
1.51
0.80
S5
24.97
2.00
1.96
S6
38.56
1.81
0.83
S7
37.37
2.01
2.36
S8
38.06
1.89
2.24
S9
36.98
1.95
1.96
S10
22.15
1.89
1.51
J1
28.88
1.93
2.03
J2
45.25
2.05
2.04
J3
40.32
2.01
1.93
J4
30.54
2.08
2.01
J5
30.48
2.02
2.04
J6
15.95
2.05
2.23
J7
25.94
2.05
2.11
J8
38.19
1.97
2.25
J9
35.54
2.10
2.08
J10
37.34
2.04
1.92
J11
31.71
2.00
1.75
J12
33.17
1.98
2.20
J13
45.25
2.11
2.11
J14
34.99
2.06
2.15
J15
32.37
2.03
2.16
J16
32.67
1.89
2.23
J17
55.52
2.09
2.11
J18
39.4
2.00
2.06
J19
26.96
1.93
2.16
J20
34.16
2.05
2.17
Restriction Digest for Plasmid Isolates
500 ng of each plasmid was used for restriction digestion with XbaI and BsaI. Two bands were observed in both correct and incorrect assemblies, allowing distinction between the two cases.
Gibson Assembly without Transformation
Gibson Assembly was performed in pBAD18 with all enzymes. The required volumes are listed in the table below.
Amounts for Gibson Assembly
Sample
Amount for Gibson [ng]
Volume Sample [µl]
Water
NEBuilder
Mut
110,5
3,44
5,76
10
Dsp B
103,5
2,29
6,91
10
Dex 1
82,09
0,83
7,68
10
Dex 2
68,66
0,69
HIs4
109,5
3,32
5,88
10
pBAD18
75
0,8
Restriction Digest Analysis for Gibson BB41 Samples
Theoretical expectation:
Preparation of 1% Agarose Gel for AMP BB41 Gibson Restriction Analysis
10 µl for PCR samples and 15 µl for restriction analysis were loaded onto the gel.
Suspecting Ampicillin depletion, fresh Ampicillin was added. All 30 overnight cultures showed growth, and plasmid isolation was successful. Restriction digestion with XbaI and BsaI was performed to identify the plasmids within the cells. Several samples indicated the presence of the desired Gibson plasmid. PCRs and PCR purification were successful, although the values were slightly lower than expected. These samples were later used for Gibson Assembly.
For the agarose gel simulation, incorrect plasmids were initially used. Upon reanalysis, it was determined that J1, J2, J8, J12, J13, J15, J17, J18, S1, S2, and S8 could be correct, as the bands were slightly above 3000 bp.
Date: Friday, 11.08.2023
Experiment: Transformation of Gibson and pSB1C3, pSB1A2, and pBAD18. Restriction Analysis of Gibson BB41. Sending for Sequencing.
Participants: Jessica 09:30-14:00; Sarah 09:30 - 16:30; Sumohit 09:30-17:00;
Summary:
Transformation of Gibson constructs from the previous day and empty vectors PSB1C3, and pSB1A2 to ensure sufficient material for BioBrick Assembly. Relevant samples were sent for sequencing.
An additional restriction analysis was performed on samples J3, J4, J5, J6, J7, J11, J14, J15, J16, S3, S4, S5, S6, and S7 to further validate the data. For this, 700 ng DNA was used with the restriction enzyme SpeI. If the plasmid is the correct construct, two bands should be visible in the gel, otherwise only one band.
Mixing Details for Restriction Analysis:
Sample Volume and Components
Sample
Volume Sample
Restriktion enzyme je
2.1 Buffer
Water
S3
19.18
0.50
5.00
24.82
S4
26.14
0.50
5.00
17.86
S5
28.03
0.50
5.00
15.97
S6
18.15
0.50
5.00
25.85
S7
18.73
0.50
5.00
25.27
J3
17.36
0.50
5.00
26.64
J4
22.92
0.50
5.00
21.08
J5
22.97
0.50
5.00
21.03
J6
43.89
0.50
5.00
0.11
J7
26.99
0.50
5.00
17.01
J11
22.08
0.50
5.00
21.92
J14
20.01
0.50
5.00
23.99
J15
21.62
0.50
5.00
22.38
J16
21.43
0.50
5.00
22.57
Analysis of Restriction Digest for Gibson BB41 Samples
Theoretical expectation:
Ordering scheme: Marker - Digestion with SpeI pBAD18-BB41 - Digestion with SpeI pBAD18 LV
Preparation of 1% Agarose Gel for Mutanase and Dex1 with AMP BB41 Primer and His4
10 µl loaded for PCR reactions.
Application Scheme:
Application scheme: Marker samples Digested with SpeI - Marker
Transformation of Various Empty Vectors and Gibson Constructs
Transformation was performed with the empty vectors pSB1C3, pSB1A2 at positions A1 and A5, and pBAD18 into the respective plates. Additionally, transformation was conducted with four Gibson constructs from the previous day. Negative control samples without plasmids were prepared for each plate.
The transformation was carried out as per the protocol.
Sequencing
As the results from the previous restriction analysis were inconclusive, samples S3, S4, J4, J7, and J11 were sent for sequencing. For sequencing, 2.5 µl of the sample and 2.5 µl of 10 µM primer were used and topped up to 10 µl with ddH2O.
Sample Concentration, Quantity for Sequencing, and Primer Information
Sample
Concentration
Amount for Seq
Primer
Seq ID
S3
36.5
91.25
BB41 For
EMX383
BB41 Rev
EMX378
S4
26.78
66.95
BB41 For
EMX379
BB41 Rev
EMX380
J4
30.54
76.35
BB41 For
EMX375
BB41 Rev
EMX376
J7
25.94
64.85
BB41 For
EMX377
BB41 Rev
EMX372
J11
31.71
79.28
BB41 For
EMX373
BB41 Rev
EMX374
Preparation of Pichia Pastoris Minimal Agar
See protocol for details.
Autoclaving of Glycerol, ddH20, and Agar solutions for Pichia Pastoris Minimal Medium. Autoclave had technical issues and had to be turned off.
Preparation of new and old His4 primers and His4 enzyme sequencing primers. Adding the appropriate amount of autoclaved ddH2O and placing the primer solution on the shaker for 1 hour at 20-25°C.
Discussion:
The second restriction analysis did not proceed as expected, and the data could not be interpreted. Therefore, five samples were selected and sent for sequencing.
For the Next Few Days:
Plating of the empty vectors overnight cultures
Evaluation of Gibson Assembly Transformation results
Analysis of the sequencing results
Date: Monday, 14.08.2023
Participants: Sarah H., Jana
His4 PCR with New and Old Primers
Reaction Setup:
Components
Volume [ul]
Q5 2xMM
25
fwd Primer
2.5
rev Primer
2.5
Template
1
ddH2O
19
PCR Program for His4:
PCR Programm
Step
Temperature [°C]
Dauer [sec]
Cycles
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s
3
Annealing
67 °C
30 s
4
Elongation
72 °C
80 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
PCRs with Enzyme Fragments to Insert AMP Overhangs
Reaction Setup:
Reaction Setup:
A
Volume [µl]
5x MM
Q5 2xMM
12.5
62.5
BB41-fwd Primer
1.25
6.25
BB41-rev Primer
1.25
6.25
Template
1
-
ddH2O
9
45
PCR Program for Enzyme Fragment PCRs:
PCR Program
Step
Action
Temperature
Duration
Cycles
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s
30 cycles
3
Annealing
67°C
30 s
30 cycles
4
Elongation
72 °C
His4: 80s; Mutanase: 90s; Dispersin B: 85s;
30 cycles
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Agarose Gel
Gel Extraction and PCR Clean-up
Gel extraction was performed for Mutanase and Dispersin B bands, along with PCR clean-up for His4 (with AMP Overhangs). The concentrations and ratios for the extracted DNA were measured.
Gel Extraction Elution:
Sample Concentration and Ratios
Sample
Concentration (ng/µl)
260/280
230/260
Mutanase
8.4
2.67
0.02
Dispersin B
60.9
10.72
4.46
PCR Clean-up Elution:
Sample Concentration and Ratios
Sample
Concentration (ng/µl)
260/280
230/260
His4
12.9
1.79
4.55
Colony PCR for pBAD Enzyme Fragments Gibson Assembly
Colony PCR was conducted for pBAD enzyme fragment Gibson Assembly using colonies from various plates.
Reaction Setup:
Reaction Setup
A
Volume [µl]
15x MM
Q5 2xMM
12.5
187.5
BB41-fwd Primer
1.25
18.75
BB41-rev Primer
1.25
18.75
Template
1 Colony
-
ddH2O
10
150
PCR Program for Colony PCR:
PCR Program
Step
Temperature
Duration
Cycles
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s
30 cycles
3
Annealing
67°C
30 s
30 cycles
4
Elongation
72 °C
90 s
30 cycles
6
Final Extension
72 °C
2 min
7
Hold
4 °C
Agarose Gel:
Overnight Cultures of pSB1C3 Vector
Three overnight cultures were prepared using 5ml LB medium and 5 ul Com(?) antibiotic for each.
Date: Tuesday, 15.08.2023
Participants: Jessica, Sarah H.
Experiment: Repeating AMP Overhang PCRs and His4 PCRs with New Primers from 14.08. with 5ng Template
His4 PCR
Reaction Setup:
Components
Volume [ul]
Q5 2xMM
25
fwd Primer
2.5
rev Primer
2.5
Template
1
ddH2O
19
PCR Program for His4:
Step
Temperature [°C]
Duration [s]
Cycles
Initial Denaturation
98
30
-
Denaturation
98
8
35 cycles
Annealing
67
30
35 cycles
Elongation
72
80
35 cycles
Final Extension
72
2 min
-
Hold
4
-
-
AMP Overhang PCRs
Reaction Setup:
A
Volume [µl]
4x MM
Q5 2xMM
25
100
BB41-fwd Primer
2.5
10
BB41-rev Primer
2.5
10
Template
1
-
ddH2O
19
76
PCR Program for AMP Overhang PCRs:
Step
Temperature
Duration
Cycles
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s
3
Annealing
69°C
30 s
4
Elongation
72 °C
90 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
-
Task3: 1% Agarose Gel for PCRs
10 ul of each PCR sample was loaded onto the agarose gel.
Task4: PCR Clean-up of His4 and Enzyme Fragments with AMP Overhangs
Gel extraction was performed for His4 and enzyme fragments with AMP Overhangs, and eluted in 10 ul.
Gel Extraction Elution:
Sample
Concentration [ng/µl]
260/280
260/230
His4 new
56.19
1.84
1.99
Miscellaneous
Pipette tips and Eppendorf tubes at IBVT were autoclaved, and ddH2O and glycerol were prepared for autoclaving. Started organizing the refrigerator compartment categorically.
Date: Wednesday, 16.08.2023
Date: Thursday, 17.08.2023
Experiment: Colony PCR, ÜN Cultures, Restriction Digest for BioBrick Assembly, PCR for Gibson Assembly of Enzyme Fragments in Pichia Vector, His4 PCR for Gibson Assembly, Linkage PCR of Dextranase
For Colony-PCR, the samples were heated at 95°C for 5 minutes before conducting the Colony PCR. ÜN cultures were prepared from picked colonies of pBAD18-enzyme fragments. PCR for Gibson Assembly of enzyme fragments in the Pichia vector was performed for all fragments except His4, followed by purification of the samples post-PCR. Restriction digestion was performed on the purified PCR samples for BioBrick Assembly. His4 PCR for Gibson Assembly of enzyme fragments in the Pichia vector was conducted. Linkage PCR for Dextranase was also carried out.
Colony-PCR
Since the previous day's colony PCR did not work, it was observed how Colony PCR is conducted, and online suggestions were found to heat the samples at 95°C for 5 minutes to denature the cells before the PCR. Mutanase primers were used for Mutanase and Dispersin B, following the Mutanase PCR program. For Dextranase, Mutanase primers were used along with Dex1 Primer and Dex2 Primer for Linkage PCR.
Mutanase and Dispersin B PCR Program (Mutanase68):
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
Linkage PCR for Dextranase (Dex):
Dex
Dex
Dex
Dex
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s
3
Annealing
68 °C
30 s
4
Elongation
72 °C
120 s
6
Final Extension
72 °C
2 min
7
Hold
4 °C
PCR for Gibson Assembly of Enzyme Fragments in Pichia Vector
Since the old PCRs with Mutanase primers or Mutanase primers – Dex1 Primer/Dex2 Primer were not found, 50 µl setups were prepared twice. Mutanase primers were used for Mut and Dsp B, Mut For Primer and Dex 1 Rev Primer were used for Dex 1, Dex2 For Primer and Mut Rev Primer for Dex 2, and Pichia primers for the Pichia vector. After PCR, 10 µl of the samples were loaded, and the remaining 40 µl were used for PCR purification.
Preparation of 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, Dex1, Dex2, and Pichia Vector
Complete 10 µl was loaded from the PCR for Gibson Assembly and 25 µl from the Colony-PCR samples.
Overnight cultures were made from pBAD18-enzyme fragment colonies and incubated at 37°C overnight.
PCR Purification and Restriction Digest for BioBrick Assembly
After PCR purification, the purified samples were used for restriction digestion. The digestion was carried out as per the NEB BioBrick Protocol. For the digestion, 500 ng of Mutanase, Dispersin B, and Pichia vector were taken, and 400 ng of His4.
Concentration Analysis (ng/µl) and Ratios
Sample
Concentration [ng/ul]
260/280
260/230
Mutanase 1
109.83
1.9
2.26
Mutanase 2
88.5
1.9
2.34
DspB1
72.87
1.89
2.17
DspB2
33.66
1.7
1.01
Dex1-1
204.1
1.93
2.37
Dex1-2
205.1
1.92
2.26
Dex2-1
127.5
1.92
2.33
Dex2-2
139.3
1.9
2.18
pichia Vektor-1
63.1
1.74
1.07
pichia Vektor-2
113.5
1.79
1.97
Volumes Used for BioBrick Assembly Restriction Digest
Sample
Volume of Sample [µl]
Restriction Enzyme 1 [µl]
Restriction Enzyme 2 [µl]
Buffer 2.1 [µl]
Water [µl]
Mutanase
4.55
EcoRI-HF
SpeI
5
38.45
Dispersin B
6.86
EcoRI-HF
SpeI
5
36.14
His4 Over
10
XbaI
PstI
5
33
pSB1C3
3.86
EcoRI-HF
PstI
5
39.14
His4 PCR for Gibson Assembly
Due to insufficient quantity of His4 for Gibson Assembly, His4 PCR was performed 5 times with the newly designed His4 primers, and 10 µl of each was loaded onto the gel.
Preparation of 1.0% Agarose Gel for His4 and pBAD18-His4 Gibson Colony-PCR
Complete 10 µl was loaded from the PCR for Gibson Assembly.
Since Dextranase is divided into two fragments, they need to be combined as one fragment. There are two methods for this, 1. Gibson Assembly and 2. Linkage PCR. Linkage PCR was chosen as it is more cost-effective than Gibson Assembly. For the Linkage PCR, the PCR-purified Dex1 and Dex2 fragments were taken, and PCR was performed with 5 ng DNA using Mutanase For and Rev Primer.
Linkage PCR Dex
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30 cycles
Annealing
68
30
30 cycles
Elongation
72
120
30 cycles
Final Extension
72
2 min
Hold
4
Discussion:
Unfortunately, the Colony PCR did not work, even though the simultaneously run PCR for Gibson Assembly did. Lysing the cells before PCR might be necessary for successful PCR. The His4 PCRs did not work, although they had worked in previous days; the reason remains unknown.
Next Steps:
Plasmid isolation from the overnight cultures and restriction analysis
His4 PCR
Gibson Assembly and BioBrick Assembly
Date: Friday, 18.08.2023
Participants: Sarah H (09:30:00-14:00); Sumohit (10:00-20:00);
Summary:
PCR amplification of His4 with old and new His4 for Gibson Assembly. Since colony PCR yielded no results, plasmids were isolated from overnight cultures and analyzed through restriction digestion. Gibson Assembly of Mutanase into the Pichia vector and BioBrick Assembly of Mutanase and Dispersin B into pSB1C3 with transformation.
His4 PCR
Since the PCR for His4 from the previous day did not work, the PCR for His4 was repeated using old and new His4 fragments and His4 primers. 10 µl of the PCR product was loaded onto the gel. The gel also included the Linkage PCR Sample from the previous day.
Creation of a 1.0% Agarose Gel for His4 and Linkage PCR
Complete 10 µl was loaded from the PCR for Gibson Assembly.
Since the Colony PCR from the previous day did not work, the plasmids were isolated from the ÜN cultures and analyzed with PstI restriction digest. Cells were grown in all samples and plasmid was isolated from the cells as in the protocol.
Sample
Concentration [ng/µl]
A260/A280
A260/A230
Mut1
23.48
2.02
2.34
Mut2
27.5
1.87
2.19
Mut3
27.61
1.91
2.12
Mut4
20.05
2.07
2.29
Mut5
20.25
1.92
2.14
DspB1
21.61
2.18
2.12
DspB2
36.03
2.17
2.26
DspB3
21.61
2.09
2.42
HIs4-1
27.34
2.13
2.29
HIs4-2
22.42
1.99
2.18
HIs4-3
27.43
2.03
2.10
Dex1
26.94
1.98
2.29
Dex2
39.56
2.02
2.21
Dex3
29.42
1.98
1.67
After plasmid isolation, 500 ng of plasmid was used for restriction digestion with PstI. If the plasmids are cloned correctly or incorrectly, two bands can be seen, which are different in both approaches and thus it can be decided which situation prevails. The table below shows how they were all mixed together. After analysis, the samples Mutanase Colony 3, Dextranase Colony 3 and His4 Colony 3 were sent for sequencing.
Sample
Volume of Sample [µl]
Restriction Enzyme PstI [µl]
Buffer 2.1 [µl]
Water [µl]
Mut1
17.04
1.00
5.00
25.96
Mut2
14.55
1.00
5.00
28.45
Mut3
14.49
1.00
5.00
28.51
Mut4
19.95
1.00
5.00
23.05
Mut5
19.75
1.00
5.00
23.25
DspB1
18.51
1.00
5.00
24.49
DspB2
11.10
1.00
5.00
31.90
DspB3
18.51
1.00
5.00
24.49
HIs4-1
14.63
1.00
5.00
28.37
HIs4-2
17.84
1.00
5.00
25.16
HIs4-3
14.58
1.00
5.00
28.42
Dex1
14.85
1.00
5.00
28.15
Dex2
10.11
1.00
5.00
32.89
Dex3
13.60
1.00
5.00
29.40
Colony PCR Analysis from Gibson Assembly and BioBrick Assembly
Analysis of restriction digestion of the Gibson Enzyme samples
theoretical expectation
Assignment scheme: Marker- PstI with pBAD18 mutanase - PstI with pBAD18 dispersin B - PstI with pBAD18-His4 - PstI with pBAD18 dextranase - PstI with pBAD18 LV - Marker
Preparation of a 1% agarose gel for restriction analysis in Gibson. 25 µl applied during the restriction analysis.
Agarose gel preparation
Prepared a 1% agarose gel for restriction analysis. Applied 25 µl during the restriction analysis.
Application scheme
Application scheme: Marker- PstI with pBAD18-mutanase (5x) - PstI with pBAD18-dispersin B (3x) - PstI with pBAD18-His4 (3x) - PstI with pBAD18-dextranase (3x) - Marker
Gibson Assembly of Mutanase in Pichia vector
Performed Gibson Assembly of Mutanase in the Pichia vector. The required volumes are provided in the table below and followed the protocol accordingly.
Sample
Amount for Gibson [ng]
Volume of Sample [µl]
Water
NEBuilder
Mut
220
2
2.07
10
HIs4
295.9
5.27
10
Pichia Vector
75
0.66
BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector
BioBrick assembly was made with mutanase and dispersin B in pBAD18 as stated in the NEB protocol.
Transformation of empty vectors and Gibson/BioBrick constructs
One transformation each was made from the empty vector pichia vector in the corresponding plates, as well as from the Gibson construct and two BioBrick Assembly constructs. For this purpose, a negative sample was made with cells without plasmids, in the Amp plate.
The transformation was performed as in the protocol
Sequencing
Since the results from the previous restriction are inconclusive the samples mutanase colony 3, dextranase colony 3 and His4 colony 3 were sent for sequencing. For sequencing, 2.5 µl of sample and 2.5 µl of 10 µM primer were used and made up to 10 µl with ddH2O.
Preparation of Pichia minimal agar selection plates.
See protocol
The agar solution was heated in a microwave and cooled to below 60°C. Then YNB, biotin and glucose solutions were added according to protocol. Plates are in cold storage of IBVT; only one agar solution a 400ml was used up the 2nd still exists in case you need to pour again.
Glycerol stock Pichia Pastoris GS115
See protocol.
Culture with YPD+His medium
Discussion:
Unfortunately, the His4 PCR did not succeed again. The Linkage PCR did show the desired PCR band, but it was very weak, and with Dex1 and Dex2 bands, which were also very weak. Plasmid isolation worked well for all samples. The restriction analysis shows mixed results.
Tasks for the Next Days:
Analyze the colonies from Gibson Assembly and BioBrick Assembly.
Analyze the sequencing results.
Date: Monday, 21.08.2023
Experiment: Repeat Transformation of Cloning from Friday. Colony PCR of His4, Dex and dispersin B-pBAD18 colonies. His4 PCR. Gibson assembly of enzyme fragments and pichia vector. BioBrick assembly of mutanase and dispersin B with the new purified PCR. Transformer of new Gibson and BioBrick assembly. Linkage PCR of Dex and gel extraction.
Participants: Sumohit 08:00-20:00
Summary:
Check plates from Friday. Perform colony-PCR of His4, Dex, and Dispersin B-pBAD18 colonies. His4 PCR. Gibson Assembly of enzyme fragments and pichia vector. BioBrick Assembly of Mutanase and Dispersin B with the newly purified PCR products. Transformation of new Gibson and BioBrick Assemblies. Linkage PCR of Dex and gel extraction.
Summary of Friday Plates Observation
The plates from Friday did not show any colonies, not even the positive control. Hence, everything needs to be repeated.
Colony PCR
Colony PCR was attempted again using iGEM 2021 Stuttgart sequencing primers for pBAD18. For the colony PCR, all three His4 samples, all three Dex samples, Dispersin B, and pBAD18 were taken as the positive control. PCR programs that work for normal PCR were used. An annealing temperature of 66 or 67 was used.
Linkage PCR of Dextranase
Since Dextranase is divided into two fragments, Dex1 and Dex2, an attempt was made to combine these two fragments through a Linkage PCR, enabling BioBrick Assembly. A PCR was performed and loaded onto the gel. Subsequently, the Dex band at approximately 4000 bp was extracted from the gel.
His4 with Overhangs PCR
Since there was insufficient His4 available, an attempt was made to perform a PCR again for the His4 fragment with the overhangs.
Preparation of a 1.0% Agarose Gel for His4 PCR, Linkage PCR, and Colony PCR
A total of 10 µl was loaded from the PCR for Gibson Assembly.
PCR cleanup was performed on these PCR samples. The values are listed in the table below.
Sample
Concentration [ng/µl]
260/280
260/230
His4 1
24.4
1.92
2.3
His4 2
47.1
1.78
2.29
Dex
2.53
0.09
Since the concentrations were satisfactory, a Gibson Assembly was performed with Mut, Dex, and Dispersin B from one PCR with a 1:1 Fragment:Vector ratio.
Gibson Assembly
Gibson Assembly was performed as outlined in the Mutanase protocol. Gibson Assembly was carried out in a pichia vector with Mutanase, Dispersin B, and Dextranase. The table below shows the required volumes and the procedure was followed as per the protocol. Since the required DNA quantities were excessive, a 1:1 Fragment:Vector ratio was used for Gibson Assembly.
Sample
Amount for Gibson [ng]
Sample Volume [µl]
Water
NEBuilder
Mut
110
1
6.17
10
Dsp B
103.9
1.43
5.74
10
Dex1
82.4
0.41
6.22
10
Dex2
69.0
0.54
His4
98.6
2.17
pichia Vector
75
0.66
BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector
For the BioBrick, 450 ng of His4 DNA digested with XbaI and PstI were used for BioBrick Assembly. BioBrick Assembly was performed in a pichia vector with Mutanase and Dispersin B, following the NEB protocol.
Transformation of Empty Vectors and Gibson/BioBrick Constructs
The DNA constructs from BioBrick Assembly and Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation process, plated on CMR-containing plates, and served as a negative control for the transformation. The transformation was carried out as per the protocol.
Transformation of Clones from Friday
The transformations from Friday were repeated following the procedure from Friday, August 18.
Date: Monday, 21.08.2023
Experiment: Repeat Transformation of Cloning from Friday
Participants: Sumohit 08:00-20:00
Summary:
Check plates from Friday. Perform colony-PCR of His4, Dex, and Dispersin B-pBAD18 colonies. His4 PCR. Gibson Assembly of enzyme fragments and pichia vector. BioBrick Assembly of Mutanase and Dispersin B with the newly purified PCR products. Transformation of new Gibson and BioBrick Assemblies. Linkage PCR of Dex and gel extraction.
Summary of Friday Plates Observation
The plates from Friday did not show any colonies, not even the positive control. Hence, everything needs to be repeated.
Colony PCR
Colony PCR was attempted again using iGEM 2021 Stuttgart sequencing primers for pBAD18. For the colony PCR, all three His4 samples, all three Dex samples, Dispersin B, and pBAD18 were taken as the positive control. PCR programs that work for normal PCR were used. An annealing temperature of 66 or 67 was used.
Linkage PCR of Dextranase
Since Dextranase is divided into two fragments, Dex1 and Dex2, an attempt was made to combine these two fragments through a Linkage PCR, enabling BioBrick Assembly. A PCR was performed and loaded onto the gel. Subsequently, the Dex band at approximately 4000 bp was extracted from the gel.
His4 with Overhangs PCR
Since there was insufficient His4 available, an attempt was made to perform a PCR again for the His4 fragment with the overhangs.
Preparation of a 1.0% Agarose Gel for His4 PCR, Linkage PCR, and Colony PCR
A total of 10 µl was loaded from the PCR for Gibson Assembly.
PCR cleanup was performed on these PCR samples. The values are listed in the table below.
Sample
Concentration [ng/µl]
260/280
260/230
His4 1
24.4
1.92
2.3
His4 2
47.1
1.78
2.29
Dex
2.53
0.09
Since the concentrations were satisfactory, a Gibson Assembly was performed with Mut, Dex, and Dispersin B from one PCR with a 1:1 Fragment:Vector ratio.
Gibson Assembly
Gibson Assembly was performed as outlined in the Mutanase protocol. Gibson Assembly was carried out in a pichia vector with Mutanase, Dispersin B, and Dextranase. The table below shows the required volumes and the procedure was followed as per the protocol. Since the required DNA quantities were excessive, a 1:1 Fragment:Vector ratio was used for Gibson Assembly.
Sample
Amount for Gibson [ng]
Sample Volume [µl]
Water
NEBuilder
Mut
110
1
6.17
10
Dsp B
103.9
1.43
5.74
10
Dex1
82.4
0.41
6.22
10
Dex2
69.0
0.54
His4
98.6
2.17
pichia Vector
75
0.66
BioBrick Assembly of Mutanase and Dispersin B in pBAD18 Vector
For the BioBrick, 450 ng of His4 DNA digested with XbaI and PstI were used for BioBrick Assembly. BioBrick Assembly was performed in a pichia vector with Mutanase and Dispersin B, following the NEB protocol.
Transformation of Empty Vectors and Gibson/BioBrick Constructs
The DNA constructs from BioBrick Assembly and Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation process, plated on CMR-containing plates, and served as a negative control for the transformation. The transformation was carried out as per the protocol.
Transformation of Clones from Friday
The transformations from Friday were repeated following the procedure from Friday, August 18.
Date: Tuesday, 22.08.2023
Experiment: Media Preparation, Overnight Cultures for Plasmid Isolation, Dex Linkage PCR, Sequencing, Colony PCR
Participants: Jana 09:00-18:00; Sumohit 09:00-18:00;
Summary:
LB medium with Amp, TB medium, and LB Agar with Amp and Cmr were prepared. Overnight (ÜN) cultures of the Gibson enzyme fragments in the pichia vector were created. Dex Linkage PCR was repeated. Colony PCR was performed with 10 minutes of boiling. Sequencing of Dex C2 was sent.
Observation of Plates from Monday
The plates from Monday showed all colonies except the BioBrick samples from Friday and the Mutanase-pichia vector 1:2. ÜN cultures were also prepared with 4 or 3 colonies per plate, so that ÜN cultures can be made the next day.
Media
Media were prepared and autoclaved as per the protocols.
His4 PCR
Performed as on Monday, August 21, 2023.
Preparation of 1.0% Agarose Gel for His4
10 µl was loaded from the PCR samples.
Loading Scheme
Marker - His 4 - His4 - Marker
PCR Purification from His4
PCR purification was performed on these PCR samples. Since the first and second His4 PCR did not show intense bands, the samples were pooled and purified. The values are listed in the table below.
Sample
Concentration [ng/µl]
260/280
260/230
His4 12
95.4
1.85
2.22
His4 3
60.0
1.86
2.48
His4 4
82.2
1.80
2.08
Linkage PCR Dex
A PCR was performed from the gel-extracted sample using Mutanase primers.
Colony PCR with Heating
Since the colony PCR from the previous day did not work, colony PCR was again performed with J1, J11, and S1. This time the samples were heated for 10 minutes at 98°C before PCR, and then PCR was performed. pBAD18 was used as a positive control, and AMP primers were used as primers.
Loading Scheme
Marker - J1 - J11 - S1 - Marker
Sequencing
For sequencing, pBAD18-Mutanase colony 4 was sent with Mutanase Forward and Reverse primers, as well as pBAD18-Dextranase with pBAD18 seq Primers Forward and Reverse.
Discussion:
The plates from Monday showed all colonies except the negative plate, Mut BioBrick old, and DspB BioBrick old. It was expected on the negative plate since they had small selection markers and were plated on selection plates. However, with Mut BioBrick old and DspB BioBrick old plates, colonies were expected, but none were visible. His4 worked well, and PCR purification was also good. Due to time constraints, no gel was made for Linkage Dex. The colony PCR with 10 minutes of heating at 98°C did not work.
Date: Wednesday, 23.08.2023
Experiment: Quick Analysis of Gibson Plasmids. Plasmid Isolation of Gibson Plasmids. Colony PCR with Lysozyme
Participants: Jana 09:00-18:00; Sumohit 09:00-18:00
Summary:
Quick analysis of Gibson plasmids. Plasmid isolation of Gibson plasmids. Colony PCR with 10 minutes of lysozyme treatment.
Quick Analysis of Gibson Plasmids
A small amount of cells was taken from the backup plates and dissolved in 15 µl of sterile ddH2O. Then proceeded as per the protocol.
A master mix of 2x NSEB solution was prepared, totaling 500 µl. After 1 hour of incubation, the cells were mixed with 25% Ficoll and applied to the gel.
Preparation of 1.0% Agarose Gel for Plasmid Quick Analysis
Completely loaded 25 µl of the samples.
Loading Scheme
pBAD18 (negative control) - Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2
The gel ran overnight at 15 V without Midori Green.
Plasmid Isolation from Gibson Colonies
Plasmids were isolated from the ÜN cultures according to the protocol. The DNA concentrations in the plasmid isolation samples are listed in the table below.
Sample
Concentration [ng/µl]
A260/A280
A260/A230
Mut 1
61.4
1.82
1.13
Mut 2
51.6
1.92
2.55
Mut 3
61.9
1.91
2.16
Disp 1
47.8
1.96
2.45
Disp 2
48.4
2.01
2.16
Disp 3
61.1
1.90
1.48
Mut 1:1 1
32.1
2.07
2.29
Mut 1:1 2
18.1
2.01
2.03
Mut 1:1 3
27.0
2.09
2.41
Mut 1:1 4
26.5
2.26
2.76
Dex 1:1 1
48.0
1.78
0.94
Dex 1:1 2
53.1
1.93
1.85
Dex 1:1 3
40.8
2.00
1.48
Dex 1:1 4
33.7
2.11
1.93
Disp 1:1 1
56.7
1.91
1.41
Disp 1:1 2
35.4
2.00
1.96
Disp 1:1 3
40.5
1.99
1.52
Disp 1:1 4
40.9
1.74
0.99
Mut C4
60.3
1.96
1.53
Colony PCR with Lysozyme
Since the colony PCR did not work, a colony PCR was performed, where initially 15 µl of lysozyme solution was added to the PCR tubes. Then cells were added, and the sample was incubated at 37°C for 10 minutes. After incubation, a regular PCR was performed with AMP primers. J1, J2, and J12 were used as samples, and AMP Fragment BB41 was taken as a positive control for PCR.
AMP BB41 Pre-experiment
PCR Program: AMPOVERH (PCR machine iGEM)
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
30
Elongation
72
18
30
Final Extension
72
120
Hold
4
forever
Discussion:
The plasmid isolation worked well, and the concentrations are above the target value.
Date: Thursday, 24.08.2023
Experiment: Restriction Analysis of Gibson Plasmids. Post-staining of Quick Analysis Gel of Gibson Plasmids.
Restriction analysis of Gibson plasmids using PstI. Post-staining with Midori Green of the quick analysis gel of Gibson plasmids.
Post-staining of Quick Analysis Gel of Gibson Plasmids
Since the gel ran overnight without Midori Green, in the morning the gel was placed in a Midori Green bath with 200 ml of Midori Green solution, which contains 40µl of Midori, and shaken for 1 hour. Then Midori Green was collected, and the gel was placed in water twice for 30 minutes each, with shaking.
Loading Scheme
pBAD18 (negative control) - Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - empty - pESP - pREP2
Restriction Analysis of Gibson Plasmids with PstI
Restriction digestion with PstI was performed on the plasmid-isolated samples. 500 ng of plasmid DNA was taken and digested with 1 µl of PstI. If the plasmids are cloned correctly, two bands are expected to be seen in all Gibson constructs, and if cloning is unsuccessful, only one band will be visible. The samples from the colony PCR from the previous day and the linkage PCR from Tuesday were also loaded on the gel.
Sample
Concentration
A260/A280
A260/A230
Mut 1
23.48
2.02
2.34
Mut 2
27.5
1.87
2.19
Mut 3
27.61
1.91
2.12
Disp 1
47.8
1.96
2.45
Disp 2
48.4
2.01
2.16
Disp 3
8.18
1.00
5.00
34.82
Mut 1:1 1
15.58
1.00
5.00
27.42
Mut 1:1 2
27.62
1.00
5.00
15.38
Mut 1:1 3
18.52
1.00
5.00
24.48
Mut 1:1 4
18.87
1.00
5.00
24.13
Dex 1:1 1
10.42
1.00
5.00
32.58
Dex 1:1 2
9.42
1.00
5.00
33.58
Dex 1:1 3
12.25
1.00
5.00
30.75
Dex 1:1 4
14.84
1.00
5.00
28.16
Disp 1:1 1
12.35
1.00
5.00
30.65
Disp 1:1 2
14.12
1.00
5.00
28.88
Disp 1:1 3
12.35
1.00
5.00
30.65
Disp 1:1 4
12.22
1.00
5.00
30.78
pBAD18
7.59
1.00
5.00
35.41
Analysis of Restriction Digestion of Gibson Enzyme Samples
Theoretical Expectation
Loading Scheme
Marker - PstI with pichia - PstI with pichia-Mutanase - PstI with pichia-Dex - PstI with pSB1C3-BBa_I20270 - PstI with pSB1C3-Mut - PstI with pSB1C3-Dispersin B - PstI with pSB1C3-Dextranase
Preparation of 1% Agarose Gel for Restriction Analysis of Gibson
25 µl was loaded for restriction analysis and other samples as well.
Below: Marker - Colony-PCR - Positive Control - Marker - Dextranase Linkage PCR with Dex 1 and Dex2 - Dextranase Linkage PCR with Dex Linkage from Gel Extraction - Marker
Discussion:
The gel from the quick analysis of Gibson plasmids is not evaluable and must be repeated on Friday. The gel from the restriction analysis also looks bad, and the gel electrophoresis with the restriction enzymes, colony PCR with lysozyme, and Dex linkage PCR need to be repeated.
Date: Friday, 25.08.2023
Experiment: Restriction Analysis of Gibson Plasmids. Quick Analysis Gel of Gibson Plasmids. Colony PCR with Lysozyme and Heating. Linkage PCR. Transformation in Pichia Cells. Sequencing of Samples.
Restriction analysis of Gibson plasmids using PstI. Post-staining with Midori Green of the quick analysis gel of Gibson plasmids.
Gel for Restriction Samples from the Previous Day
Since the gel did not run well, the gel for restriction analysis was repeated.
Analysis of Restriction Digestion of Gibson Enzyme Samples
Theoretical Expectation
Loading Scheme
Marker- PstI with Pichia - PstI with Pichia-Mutanase - PstI with Pichia-Dex - PstI with pSB1C3-BBa_I20270 - PstI with pSB1C3-Mut - PstI with pSB1C3-Dispersin B - PstI with pSB1C3-Dextranase - PstI with pBAD18 LV
Preparation of 1% Agarose Gel for Restriction Analysis of Gibson
25 µl was loaded for the restriction analysis.
Loading Scheme
Above: Mutanase BioBrick new - DspB BioBrick new - Mutanase 1:1 Gibson - Dispersin B 1:1 Gibson - Dextranase 1:1 Gibson - Marker - Marker - pBAD18 (negative control)
Repeating Gel for Quick Analysis of Gibson and BioBrick Plasmids
Since the quick analysis from previous days was not successful, it was repeated for all samples.
Sample
Concentration
A260/A280
A260/A230
Mut 1
23.48
2.02
2.34
Mut 2
27.5
1.87
2.19
Mut 3
27.61
1.91
2.12
Disp 1
47.8
1.96
2.45
Disp 2
48.4
2.01
2.16
Disp 3
8.18
1.00
5.00
34.82
Mut 1:1 1
15.58
1.00
5.00
27.42
Mut 1:1 2
27.62
1.00
5.00
15.38
Mut 1:1 3
18.52
1.00
5.00
24.48
Mut 1:1 4
18.87
1.00
5.00
24.13
Dex 1:1 1
10.42
1.00
5.00
32.58
Dex 1:1 2
9.42
1.00
5.00
33.58
Dex 1:1 3
12.25
1.00
5.00
30.75
Dex 1:1 4
14.84
1.00
5.00
28.16
Disp 1:1 1
12.35
1.00
5.00
30.65
Disp 1:1 2
14.12
1.00
5.00
28.88
Disp 1:1 3
12.35
1.00
5.00
30.65
Disp 1:1 4
12.22
1.00
5.00
30.78
pBAD18
7.59
1.00
5.00
35.41
Z cells were taken from the backup plates and dissolved in 15 µl of sterile ddH2O. Then proceeded as per the protocol.
Repeating Gel for Restriction Analysis of Gibson and BioBrick Assembly
25 µl was loaded for the restriction analysis and other samples.
Since the Dispersin B 1:1 Gibson constructs Sample 1 looked correct, it was sent for sequencing. Additionally, transformation in Pichia cells was performed with the samples Dispersin B 1:1 Gibson constructs Samples 1 and 2 following the EZ yeast transformation kit protocol and incubated at RT and 30°C over the weekend.
Date: Monday, 28.08.2023
Experiment: Colony PCR with Lysozyme and Different Cycles. Restriction Analysis. Observing Pichia Plates. Fluorescence Microscopy. BioBrick Assembly with Transformation. Quick Analysis.
Observing plates from Friday. Colony PCR with Lysozyme at 40 and 45 cycles. Examining the restriction samples from Friday. Fluorescence microscopy with BioBrick samples. New BioBrick Assembly of Mutanase and Dispersin B.
Observing Plates from Friday
On the pichia selection plates at 30°C, a few small colonies of Dsp B can be seen, but not on the others.
Colony PCR
Since slight bands were visible in the colony PCR from last week with Lysozyme, two colony PCRs were repeated for S1, J1, and J2 with 40 cycles and 45 cycles.
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
18
40/45
Final Extension
72
120
Hold
4
for ever
Restriction Analysis of BioBrick Samples
The restriction samples from Friday for BioBrick Assembly, which were not loaded onto the gel, were loaded onto the gel. 500 ng DNA was taken for the restriction digestion.
Fluorescence Microscopy
First, we were instructed by Martina on fluorescence microscopy. Then, the BioBrick samples were examined on the fluorescence microscope for GFP.
Positiv controll GFP
Negativ controll GFP
Mut1
Mut2
Mut3
Dispersin B1
Dispersin B2
Dispersin B3
BioBrick Assembly
GFP fragment bands or signals were observed on the gel and fluorescence microscope. After reviewing old posts, it was determined that the pSB1C3 restriction digestion with EcoRI and PstI needs to be purified. Therefore, the BioBrick Assembly was repeated. For this, pSB1C3 was digested again with EcoRI and PstI.
Restriction Digestion of pSB1C3
The correct band was cut and extracted from the gel.
Sample
Concentration [ng/ul]
260/280
260/230
pSB1C3
16.8
1.71
1.65
pSB1C3
20.2
1.68
0.05
Transformation of Empty Vectors and BioBrick Constructs
The DNA constructs from BioBrick Assembly were transformed into E. coli cells and plated on respective antibiotic plates. pSB1C3 was used as a positive control for the transformation and plated on CMR-containing plates. Cells without plasmids were treated as a blank in the transformation, plated on CMR-containing plates, and served as a negative control for the transformation.
The transformation was carried out as per the protocol.
Date: Tuesday, 29.08.2023
Experiment: Restriction Digestion of pBAD18 Enzyme Fragments. Restriction Analysis of pichia Vector Enzyme Fragments. UV Irradiation. Fluorescence Microscopy. Setting up Overnight Cultures. AMP Cloning.
Restriction digestion of pBAD18 enzyme fragments. Observing plates from Friday. Restriction digestion of pichia vector enzyme fragments to detect the fragments. UV irradiation. Fluorescence microscopy. Setting up overnight cultures. Gibson Assembly of AMP1 and AMP2 into pBAD18.
Observing Plates from Friday
On the pichia selection plates at 30°C and RT, a few small colonies of Dsp B can be seen, but not on the others.
Restriction Digestion of pBAD18 Enzyme Fragments
Since hardly any colonies were visible on the plates by tomorrow 10 AM and there was growth in the positive control, it was assumed that the restriction digestion of the Gibson overhangs is not sufficient for BioBrick Assembly. Therefore, pBAD18-Mut and -Dsp B were digested with EcoRI and SpeI, and pBAd18-His4 was digested with XbaI and PstI so that BioBrick Assembly can be done with these samples. After digestion, the samples were loaded onto the gel.
Restriction Digestion of pichia Enzyme Fragments
Since so far only the His4 fragment could be detected on the gels, a new restriction analysis with EcoRI and PstI was performed so that the upper band in the previous gels can separate into the enzyme fragment band and backbone band. 500 ng DNA was digested with EcoRI and PstI.
PCR for AMP1 and AMP2 Gibson
The AMP1 and AMP2 fragments were diluted in water to achieve an end mass concentration of 10ng/µl and then a PCR was performed with them.
PCR programm for AMP1
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
18
40/45
Final Extension
72
120
Hold
4
for ever
PCR programm for AMP2
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
40
40/45
Final Extension
72
120
Hold
4
for ever
Creating a 1.0% Agarose Gel for Restriction Digestion and AMP PCR
25 µl was loaded from the restriction digestion and PCR samples onto the gel.
UV Irradiation of BioBrick Cells
Cells have GFP if the colonization was not successful. Therefore, the cells were examined under a UV lamp in Mibi, and colonies 4, 15, and 17 from Mutanase BioBrick plate and colonies 14 and 21 from Dextranase plate did not show any or very weak signals. Therefore, these samples were taken and examined under a fluorescence microscope.
Fluorescence Microscopy of BioBrick Cells
The cells that showed no or very weak signal were examined under the fluorescence microscope and GFP was observed in the cells.
AMP1 and AMP2 Gibson Assembly
Due to communication issues during sample loading in the previous gels, the entire PCR approach was loaded onto the gel. Therefore, the PCR for AMP1 and AMP2 was repeated, and for AMP2, two PCR approaches with 50 µl each were made.
PCR programm for AMP1
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
18
40/45
Final Extension
72
120
Hold
4
for ever
PCR programm for AMP2
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
40
40/45
Final Extension
72
120
Hold
4
for ever
PCR purification was made from these PCRs. The values are listed in the table below.
Sample
Concentration [ng/ul]
260/280
260/230
AMP1
116.9
1.85
2.27
AMP2-1
93.0
1.90
2.46
AMP2-2
92.3
1.84
2.4
Quick Analysis of BioBrick Colonies
Performed as documented in the protocol for the BioBrick samples.
Preparation of a 1.0% Agarose Gel
The complete samples were loaded onto the gel.
Application Scheme
Marker
Mutanase BB
Dispersin B BB
Dextranase BB
His4 BB
pBAD18
pSB1C3
Marker
Gibson Assembly
Performed as described in the protocol. The table below shows the DNA quantities and sample volumes used for Gibson Assembly.
Sample
Amount for Gibson [ng]
Volume Sample [µl]
Water
NEBuilder
AMP1
19.63
0.1679213003
9.0320786997
10
AMP2
45.47
0.4889247312
8.7110752688
10
pBAD18 Vektor
75
0.80
Transformation of Empty Vectors and Gibson Constructs
The DNA constructs from the Gibson Assembly were transformed into E. coli cells and plated on the corresponding antibiotic plates. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank in the transformation, plated on Amp-containing plates, and served as a negative control for transformation.
The transformation was carried out as per the protocol.
Setting Up Overnight Cultures
Overnight cultures were made from all BioBrick colonies from the previous cloning. Overnight cultures were also made from the samples that showed no signal during UV irradiation and from the colonies from the Gibson Assembly of enzyme fragments into the pichia vector, which are most likely correct.
Date: Wednesday, 30.08.2023
Experiment Summary
Experiment: Plasmid Isolation and Restriction Analysis of Pichia Enzyme Fragments and BioBrick Assembly Samples. Setting up Overnight Cultures. Maintenance.
View plates from Tuesday. Perform plasmid isolation and restriction analysis of Pichia enzyme fragments and BioBrick assembly samples (old and new). Set up overnight cultures. Maintenance.
View Plates from Tuesday
Samples were visible on AMP plates as well as on the positive control.
Plasmid Isolation
Plasmids were isolated from the overnight cultures according to the protocol. The table below shows the DNA concentrations in the plasmid isolation samples.
Sample
Concentration [ng/ul]
A260/280
A260/A230
4;1
39.1
2.07
2.29
4;2
54.5
2.09
2.75
14;1
58.1
2.17
2.7
14;2
57.4
2.17
2.71
15;1
49.4
2.1
2.51
15;2
41.9
2.06
2.26
17;1
29.6
2.12
2.54
17;2
36.6
2.12
2.44
21;1
26.4
2.14
2.79
21;2
16.9
2.17
2.42
Dex2
24.3
2.49
2.69
Dex4
18.1
2.04
2.73
BB Mut1
44.5
2.13
2.45
BB Mut2
42.7
2.25
2.89
BB Mut3
43.5
1.99
2.13
BB Mut4
58
2.18
2.42
BB Mut5
24
2.22
1.92
BB Mut6
37.1
2.22
2.03
BB Dsp B 1
51
2
1.55
BB Dsp B 2
29.5
2.17
1.95
BB Dsp B 3
33.6
2.21
2.23
BB Dsp B 4
24.8
2.13
2.11
Dsp B1
42.7
2.08
1.92
Dsp B2
41.9
2.11
2.24
Restriction Analysis of Plasmids
Since the restriction analysis from the previous day did not show the desired bands, the restriction analysis was repeated with different enzymes. BsaI was added to Dispersin B samples from the previous day, and HindIII was added to Dextranase samples from the previous day. Additionally, a restriction analysis was performed with the old BioBrick samples and the BioBrick samples from the latest cloning using EcoRI and PstI.
Sample
Amount for Gibson [ng]
Volume Sample [µl]
Water
NEBuilder
4;1
12.79
2.00
5.00
28.21
4;2
9.17
2.00
5.00
31.83
14;1
8.61
2.00
5.00
32.39
14;2
8.71
2.00
5.00
32.29
15;1
10.12
2.00
5.00
30.88
15;2
11.93
2.00
5.00
29.07
17;1
16.89
2.00
5.00
24.11
17;2
13.66
2.00
5.00
27.34
21;1
18.94
2.00
5.00
22.06
21;2
29.59
2.00
5.00
11.41
Dex2
20.58
2.00
5.00
20.42
Dex4
27.62
2.00
5.00
13.38
BB Mut1
11.24
2.00
5.00
29.76
BB Mut2
11.71
2.00
5.00
29.29
BB Mut3
11.49
2.00
5.00
29.51
BB Mut4
8.62
2.00
5.00
32.38
BB Mut5
20.83
2.00
5.00
20.17
BB Mut6
13.48
2.00
5.00
27.52
BB Dsp B 1
9.80
2.00
5.00
31.20
Sample
Conc. [ng/ul]
Restriktion enzyme
Puffer 2.1
Water
BB Dsp B 2
16.95
2.00
5.00
24.05
BB Dsp B 3
14.88
2.00
5.00
26.12
BB Dsp B 4
20.16
2.00
5.00
20.84
Dsp B1
11.71
2.00
5.00
29.29
Dsp B2
11.93
2.00
5.00
29.07
Analysis of Restriction Digests
Preparation of a 1% Agarose Gel for restriction analysis of Gibson. 25 µl loaded for the restriction analysis and other samples as well.
Setting up Overnight Cultures
Set up overnight cultures from 5 colonies from the AMP1 plate, 5 colonies from the AMP2 plate, and 18 different colonies from the pichia-DspB plate, and streaked onto additional plates.
Date: Thursday, 31.08.2023
Experiment Summary
Experiment: Plasmid Isolation and Restriction Digest. Samples sent for sequencing. Quick analysis. pSB1C3 Restriction Digest and Gel Extraction.
Plasmid Isolation and Restriction Analysis of AMP1 and AMP2 colonies. Quick analysis of AMP1 and AMP2 colonies. pSB1C3 Restriction Digest and Gel Extraction. Sequencing.
Plasmid Isolation
Plasmids were isolated from the overnight cultures according to the protocol. The table below shows the DNA concentrations in the plasmid isolation samples.
Sample
Conc. [ng/ul]
A260/280
A260\\230
AMP1-1
52.6
1.94
2.3
AMP1-2
46.6
2.0
2.39
AMP1-3
8.9
1.76
2.16
AMP1-4
79.7
1.98
1.99
AMP1-5
62.9
2.02
2.28
Mut-4
16.1
1.9
2.08
Kontrolle
19.2
1.96
1.72
AMP2-1
45.2
2.0
2.4
AMP2-2
36.5
1.94
2.14
AMP2-3
25.1
1.98
1.89
AMP2-4
58.4
2.0
2.16
AMP2-5
58.9
2.02
2.34
Restriction Analysis of Gibson Plasmids with PstI
The AMP samples were digested with PstI for the restriction analysis. 1000 ng of plasmid DNA was taken and digested with 1 µl PstI. If the plasmids are properly cloned, two bands can be seen in all Gibson constructs, and if the cloning was unsuccessful, only one band will be visible. Quick analysis samples for AMP Gibson were also loaded onto the gel. Since the gel ran overnight, 5 µl of all AMP samples were loaded onto a small gel so that some samples can be sent for sequencing.
Sample
Conc. [ng/ul]
A260/280
Puffer 2.1
AMP1-1
52.6
1.94
2.3
AMP1-2
46.6
2.0
2.39
AMP1-3
8.9
1.76
2.16
AMP1-4
79.7
1.98
1.99
AMP1-5
62.9
2.02
2.28
Mut-4
16.1
1.9
2.08
Kontrolle
19.2
1.96
1.72
AMP2-1
45.2
2.0
2.4
AMP2-2
36.5
1.94
2.14
AMP2-3
25.1
1.98
1.89
AMP2-4
58.4
2.0
2.16
AMP2-5
58.9
2.02
2.34
Analysis of Restriction Digests of Gibson Enzyme Samples
Theoretical Expectations
Gel Preparation for Restriction Analysis of Gibson
25 µl were loaded for the restriction analysis and other samples in the large gel (Ready on Friday after post-staining). 5 µl were loaded for the restriction analysis in small gel.
Quick Analysis
With the latest BioBrick cloning, a quick analysis of the plasmids was performed and loaded onto the gel. Unfortunately, the negative control ran out of the well, and it was decided to repeat the entire quick analysis as the evaluation can be difficult without control. A quick analysis of a colony from pSB1C3 was also performed, serving as a negative control for the plasmid quick analysis.
Sequencing
After some samples appeared successful for cloning, a few samples were sent for sequencing.
pSB1C3 Restriction and Gel Extraction
Since the amount of pSB1C3 seemed insufficient and more was needed, 500 ng of pSB1C3-BBaI20270 was digested with EcoRI and PstI and loaded onto the gel.
Experiment: Post-staining of Quick Analysis and Restriction Analysis Gel of Gibson Plasmids. Pelleting of Pichia with Dispersin B. Pichia Transformation. Colony PCR of Pichia samples.
Participants:Sumohit 09:30-17:30
Summary
Post-staining with Midori Green of the Quick Analysis and Restriction Analysis Gel of Gibson Plasmids. The Pichia cell cultures were centrifuged and stored as pellets. Transformations were done in Pichia with Mutanase 1:1 Colony 4 and Dextranase 1:1 Colony 2 and 4. Colony PCR of Pichia with Dispersin B.
Post-staining of Quick Analysis and Restriction Analysis Gel of Gibson Plasmids
Since the gel ran overnight without Midori Green, the gel was placed in a Midori Green bath in the morning with 200 ml Midori Green solution, containing 40µl of Midori, and shaken for 1 hour. Then the Midori Green was collected and the gel was placed in water twice for 30 minutes each and shaken.
Ordering Scheme
Top
pBAD18 (negative control) - mutanase BioBrick new - DspB BioBrick new - mutanase 1:1 Gibson - dispersin B 1:1 Gibson - dextranase 1:1 Gibson - empty - pESP -pREP2
Bottom
Marker - pBAD18 (negative control) - Mutanase BioBrick old - DspB BioBrick old - pESP -pREP2 - Marker
Top
Pelleting of Pichia-Dispersin B Cultures
Attempts were made to pellet from the Pichia cultures. Unfortunately, it seemed like nothing had grown in the Pichia cultures, and after centrifuging at 14,000 rpm for 5 minutes, no pellet was visible.
Pichia Transformation
In the Pichia cells, according to the protocol, Mutanase 1:1 Colony 4 and Dextranase 1:1 Colonies 2 and 4 were transformed. Additionally, a few Pichia cells without transformation were streaked onto a plate, serving as a negative control for the transformation.
Colony PCR Pichia
Since the cultures were not growing, colony PCR was performed to confirm whether Dispersin B has integrated into the genome of the cells or is present in the cells. For this, an online protocol was followed where the cells were lysed with 20µl of 20 nM NaOH at 98°C for 45 minutes. The supernatant was collected after centrifugation for PCR. PCR was performed using 1 µl of the supernatant with Mutanase For and Rev Primers for confirmation. Additionally, a PCR with Dispersin B from IDT Eppi was used as a template, serving as a positive control for the PCR itself.
Mutanase and Dispersin B PCR Program: Mutanase68
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
Date: Monday, 11.09.2023
Experiment: Colony-PCR of Pichia Samples. Restriction Analysis of pBAD18-AMP1 and -AMP2. Preparing Overnight Cultures.
Participants: Sumohit 09:00-19:30
Summary: Colony-PCR of Pichia samples with 45 minutes of heating beforehand. Restriction analysis of pBAD18-AMP1 and -AMP2 with PstI. Setting up ÜN cultures for AMP1, AMP2, Dex, and Mut samples.
Colony-PCR Samples from Friday
Colony-PCR was performed on the samples from Friday and loaded onto a gel.
Preparation of 1% Agarose Gel for Colony-PCR of Pichia Dispersin B Cells
15 µl of the samples were loaded.
Application Scheme
Gel1
Marker - White Pichia Colonies - Dsp B Control PCR - Small White Pichia Colonies - Marker
Gel2
Marker - Beige Pichia Colonies - Dsp B Control PCR - Marker - Mutanase digested with SalI - DspB digested with SalI - Dextranase digested with SalI - Marker
Restriction Analysis of pBAD18-AMP1 and -AMP2 Gibson
Since the restriction analysis from last week with AMP1 and AMP2 did not show many bands and the ones shown were weak, the restriction analysis was repeated.
Sample
Conc. [ng/ul]
A260/280
A260/A230
AMP1-1
52.6
1.94
2.3
AMP1-2
46.6
2
2.39
AMP1-3
8.9
1.76
2.16
AMP1-4
79.7
1.98
1.99
AMP1-5
62.9
2.02
2.28
Mut-4
16.1
1.9
2.08
Kontrolle
19.2
1.96
1.72
AMP2-1
45.2
2
2.4
AMP2-2
36.5
1.94
2.14
AMP2-3
25.1
1.98
1.89
AMP2-4
58.4
2
2.16
AMP2-5
58.9
2.02
2.34
Analysis of Restriction Digestion of Gibson Enzyme Samples
Theoretical Expectation
Application Scheme: Marker - PstI with pBAD18-AMP2 - PstI with pBAD18-AMP1 - PstI with pBAD18 LV
Preparation of 1% Agarose Gel for Restriction Analysis of Gibson
Overnight cultures were set up for all old AMP1 and AMP2 backup colonies and 3 new colonies each. ÜN was also set up for Dex Colony 2 and Dex Colony 4 1:1 Pichia Gibson twice, and Mut Colony 4 1:1 Gibson.
Date: Tuesday, 05.09.2023
Experiment: Colony-PCR of S1 samples. Colony-PCR Disp B Pichia. Plasmid Isolation and Restriction Analysis
Participants: Sumohit 08:00-18:30
Summary: Progress of Colony-PCR of S1 samples with heating and variations. Colony-PCR of Pichia samples with Dsp B after NaOH lysis. Plasmid isolation. Restriction analysis of Mutanase and Dextranase samples.
Colony-PCR Trial
Since some bands were observed in the Pichia samples, it was decided to treat the E. coli cells in a similar manner. For the trial, S1 sample colonies were taken, known for correct cloning. For the trial, a colony was picked, dissolved in 20µl 20mM NaOH, and heated at 98°C for 45 minutes. Another colony was taken, dissolved in 20µl ddH2O, and heated at 98°C for 45 minutes. Another colony was dissolved in 20µl 20 Lysozyme and heated at 37°C for 45 minutes. As a control, 1 µl BB41 was dissolved in 20 µl 20mM NaOH to observe if it can damage the DNA. Additionally, a PCR was performed with 1 µl BB41 in water, serving as a positive control for the PCR.
AMP PCR Program: AMPOVERH
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
18
40/45
Final Extension
72
120
Hold
4
for ever
Preparation of 1% Agarose Gel for Colony-PCR Trial
15 µl of samples were loaded.
Application Scheme
Marker - Colony in NaOH - Colony in Water - Colony in Lysozyme - DNA in NaOH - AMP Positive Control - Marker
Colony-PCR Pichia-Dsp B
The colony-PCR from the previous day was repeated since not even the positive control showed the correct band size, and the Colony-PCR trial showed very good results. The cells were lysed again in 20 µl 20 NaOH and used for the PCR. A positive control for the PCR was also prepared.
Mutanase and Dispersin B PCR Program
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
Preparation of 1% Agarose Gel for Colony-PCR Pichia Dispersin B Cells
15 µl of samples were loaded.
Application Scheme
Marker - Colonies from white and small white Pichia samples - Marker - Dsp B Positive Control - Marker
Plasmid Isolation
Plasmids were isolated from the ÜN culture following the protocol. The DNA concentrations in the plasmid isolation samples are provided in the table below.
Sample
Conc. [ng/ul]
A260/280
A260/A230
AMP1-1
52.6
1.94
2.3
AMP1-2
46.6
2
2.39
AMP1-3
8.9
1.76
2.16
AMP1-4
79.7
1.98
1.99
AMP1-5
62.9
2.02
2.28
Mut-4
16.1
1.9
2.08
Kontrolle
19.2
1.96
1.72
AMP2-1
45.2
2
2.4
AMP2-2
36.5
1.94
2.14
AMP2-3
25.1
1.98
1.89
AMP2-4
58.4
2
2.16
AMP2-5
58.9
2.02
2.34
Restriction Analysis of Gibson Plasmids (Mutanase; Dispersin B; Dextranase) with EcoRI and PstI
It was suspected that the restriction analysis last week did not work for unknown reasons, so it was repeated with EcoRI and PstI for Mutanase Colony 4, Dextranase Colony 2, and Colony 4. For the restriction analysis, 500 ng of DNA was used with 1µl of restriction enzyme each.
Preparation of 1% Agarose Gel for Restriction Analysis of Gibson
25 µl were loaded for the restriction analysis.
Application Scheme
Marker - PstI with Pichia vector-Mutanase - PstI with Pichia vector-Dispersin B - PstI with Pichia vector-Dextranase - PstI with Pichia vector
Preparation of 1% Agarose Gel for Restriction Analysis of Gibson
25 µl were loaded for the restriction analysis.
Application Scheme
Marker - PstI with Pichia vector-Mutanase I - PstI with Pichia vector-Mutanase II - PstI with Pichia vector-Dispersin B I - PstI with Pichia vector-Dispersin B II - PstI with Pichia vector-Dextranase I - PstI with Pichia vector-Dextranase II - Marker
Unfortunately, there is no image. Only the His4 band was visible again along with the enzyme fragment band and the backbone band. Ideally, this band should be seen as two bands, one for the backbone and one for the enzyme fragment in the gel.
Date: Wednesday, 06.09.2023
Experiment: Colony-PCR of AMP1 and AMP2 samples; Colony-PCR of Mutanase and Dispersin B; Gibson Assembly; Media
Participants: Sarah 13:00-15:00; Sumohit 09:00-19:30
Summary: Colony-PCR of AMP1 and AMP2 samples. Colony-PCR of Mutanase and Dispersin B samples. Gibson Assembly of enzyme fragments in Pichia vector. Preparation of YPD medium.
Colony-PCR of Mutanase and Dispersin B samples for Pichia
Colony-PCR was performed for Pichia vector-Mutanase colony 4 and Dispersin B colonies 1 and 2. The Colony-PCR was conducted as per the protocol. Additionally, a positive control was included for each, with 1 µl from the respective fragment stock.
Mutanase and Dispersin B PCR Program
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
Colony-PCR of AMP1 and AMP2 samples
Colony-PCR was performed for pBAD18-AMP1 and -AMP2. The Colony-PCR was conducted as per the protocol. Additionally, a positive control was included for each, with 1 µl from the respective fragment stock.
PCR programm for AMP1
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
18
40/45
Final Extension
72
120
Hold
4
for ever
PCR programm for AMP2
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
40/45
Annealing
69
30
40/45
Elongation
72
40
40/45
Final Extension
72
120
Hold
4
for ever
Preparation of 1% Agarose Gel for Colony-PCR AMP1 Colonies
Marker - AMP2-1 - AMP2-2 - AMP2-3 - AMP2-4 - AMP2-5 - AMP2-6 - AMP2-7 - AMP2-8 - Marker - Mutanase Colony 4 - Mutanase + Control - Dispersin B Colony 1 - Dispersin B Colony 2 - Dispersin B + Control - Marker - AMP2 + Control (200 µl PCR Tube) - AMP2 + Control (500 µl PCR Tube) - AMP2 DNA in 20 µl 20 NaOH - Marker
Gibson Assembly
Since there was suspicion of errors in the cloning of enzyme fragments into the Pichia vector, the 1:1 vector:fragment Gibson Assembly was repeated. The required volumes are shown in the table below and were followed as per the protocol.
Sample
Amount for Gibson [ng]
Sample Volume [µl]
Water [µl]
NEBuilder
Mut
110
1
6.17
10
Dsp B
103.9
1.43
5.74
10
Dex1
82.4
0.41
6.22
10
Dex2
69.0
0.54
HIs4
98.6
2.17
pichia Vektor
75
0.66
Transformation of Empty Vectors and Gibson Constructs
The DNA constructs from the Gibson Assembly were transformed into E. coli cells and plated on respective antibiotic-containing plates. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank during transformation, plated on Amp-containing plates, and served as a negative control for transformation. The transformation was conducted as per the protocol.
Media
500 ml of YPD medium was prepared twice.
Date: Thursday, 07.09.2023
Experiment: Colony-PCR AMP1 and AMP2 Samples; Sequencing; Colonies Plating; Colony-PCR for Enzyme Fragments and Peptide Fragments; Genomic Isolation; Restriction Digestion of AMP Samples; GFP and AMP2 Expression
Participants: Sumohit 09:00-22:30
Summary: Gel for Colony-PCR for AMP1 and AMP2. Sending samples for sequencing. Plating colonies on backup plates. Colony-PCR for pichia vector-Mutanase, -Dextranase, -Dispersin B. Preparation for genomic isolation and setting up ÜN cultures. Restriction digestion of AMP samples. GFP and AMP2 expression.
Colony-PCR for Pichia Vector-Dextranase old Gibson Assembly
Colony PCR was performed for Dextranase Colony 2 and Colony 4 from the old Gibson Assembly for the enzyme fragments in the Pichia vector. The samples were loaded on the AMP gel.
Linkage PCR for Dextranase (Dex)
Step
Temperature [°C]
Duration [sec]
Cycles
1
Initial Denaturation
98 °C
30 s
2
Denaturation
98 °C
8 s (30 cycles)
3
Annealing
68 °C
30 s (30 cycles)
4
Elongation
72 °C
150 s (30 cycles)
6
Final Extension
72 °C
3 min
7
Hold
4 °C
Continuous
Gel for Colony PCR AMP1 and AMP2
As the bands in the gels for AMP1 and AMP2 were too smeary, we discussed this issue with MiBi and they mentioned that this happens when GelRed is used in the gel. In MiBi, they do post-staining with GelRed, and the same was done here.
Preparation of 1% Agarose Gel for Colony-PCR AMP Colonies and Dextranase Colony from old Gibson Assembly
Samples AMP1-1; AMP1-6; AMP1-7; AMP2-6; AMP2-7; and AMP2-8 were sent for sequencing using AMP forward and reverse primers. All samples look correct except AMP2-8, which, according to both primers, has a mismatch in GFP resulting in a sequence change.
Examining Plates with Gibson Assembly from Wednesday
Images were forgotten to be taken. The plates are there and will be done later. Colonies have grown everywhere as expected except in the negative control.
Colonies Plating
To avoid waiting for long, 12 colonies from each pichia enzyme fragment were plated on a backup plate and incubated at 37°C for 6 hours until enough cell material is available to perform a colony-PCR.
Colony PCR with Pichia Vector-Enzyme Fragment new Gibson Assembly
The cells from the previously plated colonies were used to perform colony PCR. The cells were dissolved in 20µl 20 mM NaOH and heated at 98°C for 45 minutes. After that...
Preparation for Genomic Isolation and overnight Cultures
5.99 g of lithium acetate was procured by Rebekka and a 200 mM lithium acetate solution in water (final volume 50 ml) was prepared. ÜN cultures from Biege 1 to 4 of the pichia cells with Dsp B were made in the self-made YPD medium and incubated overnight at 37°C.
Restriction Digestion of pBAD18-AMP1 and -AMP2 Gibson
Since the restriction analysis last week with AMP1 and AMP2 did not show many bands and the ones shown were weak, the restriction analysis was repeated. Here, 1000 ng of DNA was used and after the restriction digestion, the samples will be purified so that...
Sample
Amount [ng]
Restriktion enzyme
Puffer 2.1
Water
AMP1-1
52.6
1.94
2.3
AMP1-2
46.6
2
2.39
AMP1-3
8.9
1.76
2.16
AMP1-4
79.7
1.98
1.99
AMP1-5
62.9
2.02
2.28
Mut-4
16.1
1.9
2.08
Kontrolle
19.2
1.96
1.72
AMP2-1
45.2
2
2.4
AMP2-2
36.5
1.94
2.14
AMP2-3
25.1
1.98
1.89
AMP2-4
58.4
2
2.16
AMP2-5
58.9
2.02
2.34
GFP and AMP2 Expression
After the colony PCR and sequencing provided the desired results, 10 ml TB medium with AMP2 colonies (AMP2-3, AMP2-6, AMP2-7, AMP2-8) was inoculated. Due to time constraints, the cells were induced with 100 µl 2% arabinose at OD600 0.257; 0.452; 0.289; and 0.313 and incubated further at 37°C overnight.
Date: Friday, 08.09.2023
Experiment: Fluorescence Microscopy; Genomic Isolation; Purification of Restriction Digestion; Post-staining; Preparation for AMP Expression; Preparation for Genomic Integration; Colony-PCR; PCR
Participants: Sarah 09:30-17:00; Sumohit 10:00-19:00
Summary: Fluorescence microscopy of expression samples; Genomic isolation of pichia cells with DspB; Purification of AMP1 and AMP2 restriction digestion samples; Post-staining; Preparation for AMP expression; Preparation for genomic integration; Colony-PCR of Mutanase, Dispersin B, and Dextranase; PCR of AMP2 samples for sequencing
Post-staining
A 3% GelRed solution was prepared, and the gel from the previous day was immersed in 200 µl GelRed solution and incubated on the shaker for 20 minutes. Afterward, the gel was washed twice with water for 20 minutes and analyzed.
All AMP samples showed a GFP signal, including the AMP2-3 sample, although no signal was expected here. The saline control showed no GFP signal.
Images were taken, but it's difficult to see much in the pictures.
Genomic DNA Isolation from Pichia Cells
The genomic DNA from the ÜN culture samples from the previous day was isolated and stored as per the protocol by iGEM Team tjusls China 2022.
Lithium acetate was dissolved in water instead of acetic acid as found in all relevant papers.
Purification of AMP Restriction Samples
The AMP restriction samples from the previous day were purified using the Zymo Kit, as recommended by the manufacturer. The concentrations are listed in the table below.
Preparation for AMP Expression
Flasks were autoclaved, and the shaker in the MiBi laboratory was checked for functionality.
Colony PCR of Mutanase and Dispersin B
PCR was performed for Mutanase and Dispersin B using the lysed cells from Thursday. A 25 µl reaction was prepared with 1µL from the lysate. After PCR, the Mutanase samples were loaded on the gel. Additionally, a positive control for the PCR was made and loaded.
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
After gel electrophoresis, the gel was restained with GelRed and analyzed.
PCR of AMP2 Samples for Sequencing
Since the data from the AMP2-6 and AMP2-7 sequencing with the AMP Forward Primer did not arrive and there was not much plasmid available, a PCR was performed with AMP2-6 and AMP2-7. The samples were purified with the PCR purification kit and sent for sequencing with the AMP Forward Primer.
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
10
30
Annealing
69
30
30
Elongation
72
40
30
Final Extension
72
120
Hold
4
for ever
Sample
Concentration [ng/ul]
260/280
260/230
AMP2-6
45.9
1.90
2.46
AMP2-7
51.2
1.84
2.4
Date: Monday, 11.09.2023
Participants:Participants: Sarah H.
Summary: Making of TB Medium. Restriktion digestion. Overbnight culture.
10 min digestion at 37°C, followed by 20 min heat inactivation at 65°C.
ÜN Cultures of AMP Colonies: AMP2-6, AMP2-8, AMP1-6, and AMP1-7
5 ml TB Medium with salt + 5 ul Amp (100mg/ml)
Date: Tuesday, 12.09.2023
Participants: Sarah (09:30-17:00), Sumohit (08:00-20:30)
AMP Expression
After detecting GFP in the AMP2 samples, it was decided to start the expression of samples AMP1-6, -7, and AMP2-6, -7. For expression, 1 ml of the overnight culture was taken and added to 100 ml of TB medium. The cells were incubated at 30°C on a shaker, continuously shaking. During expression, the optical density (OD600) of the samples was continuously measured. At an OD600 of 0.4-0.6, the expression was induced by adding 1 ml of 2% arabinose solution. Expression was started at 08:30 AM, induced at 12:45 PM, and ended at 08:00 PM.
Gibson Assembly of Enzyme Fragments in pichia Vector
Was performed as per the protocol. Gibson Assembly was performed in the pichia vector with Mutanase, Dispersin B, and Dextranase. Additionally, Gibson Assembly was performed in pBAD18 with Mutanase and Dispersin B. The required volumes are listed in the table below. Since the 1:1 Gibson cloning did not yield the desired results, a 1:2 vector-to-fragment Gibson Assembly was performed for both pichia vector and pBAD18.
Sample
Amount for Gibson [ng]
Volume Sample [µl]
Water
NEBuilder
Mut
220
2
5.27
10
Dsp B
207.8
2.86
4.41
10
Dex1
164.8
0.84
5.35
10
Dex2
138.0
1.08
HIs4
197.2
2.07
pichia Vektor
75
0.66
Sample
Amount for Gibson [ng]
Volume Sample [µl]
Water
NEBuilder
Mut
110.5
1.44
6.67
10
Dsp B
101.9
1.31
6.8
10
HIs4
98.2
1.09
pBAD18 Vektor
75
0.80
Transformation of Empty Vectors and Gibson Constructs for pichia Gibson
Only the DNA constructs from the Gibson Assembly for pichia Gibson were transformed into E. coli cells and plated on the corresponding antibiotic-containing plates. Due to a shortage of LB-agar plates with Amp, not enough were available for all samples. pBAD18 was used as a positive control for transformation and plated on Amp-containing plates. Cells without plasmids were treated as blanks in the transformation, plated on Amp-containing plates, and used as a negative control for the transformation. The transformation was performed as per the protocol.
Preparation for Improvement of Existing Part
Over the past years, many teams have successfully worked with Dispersin B. Therefore, it was decided to search for plasmids in the iGEM distribution kit containing Dispersin B. In the iGEM distribution kit 2021, on Plate 5, three fragments with Dispersin B were identified, found at positions 15D, 15F, and 17D. These fragments were resuspended from the corresponding wells and transformed into E. coli. For the transformation, 1 µl of the solution was taken and transformed into the cells. The transformed solution was then plated on CMR-containing agar plates. The transformation was performed as per the protocol.
Component
Volume [ul]
14 x MM
Q5 2xMM
12.5
175
Mut fwd Primer
1.25
17.5
His4 rev neu Primer
1.25
17.5
Template
1
-
ddH2O
9
126
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
Dex: 165; Mut: 140; DispB: 140
30
Final Extension
72
Mut und Disp: 120; Dex: 300
Hold
4
forever
Gel Preparation for Colony PCRs and SalI-HF Restriction Digest (Mo)
1% agarose gel was prepared for Colony PCRs and SalI-HF restriction digest.
Centrifugation and Resuspension in Saline of E. coli Cells with Amp1 and Amp2
E. coli cultures were centrifuged at 5000g for 20 minutes and resuspended in 3ml of 0.9% saline. The samples included Amp1-6, Amp1-7, Amp2-6, Amp2-7, and 0.9% saline.
The E. coli solutions were stored in the -20°C freezer.
Sequencing of Some Minipreps from August 23rd with the Correct Sequencing Primer
1:2 dilution of Enzyme-His4 Sequencing Primer was prepared. 5 ul sample was mixed with 5 ul of the primer (1:2 dilution).
Biobrick plates are still in the incubator, all others are in the refrigerator.
ÜN Culture of E. Coli BW...
5ml TB medium with salts. E. coli picked from a plate without antibiotics. Incubation at 37°C.
Date: Thursday, 14.09.2023
Experiment Summary: Colony-PCR, GFP Analysis in AMP2, PCR Tests, Media Preparation, Gibson Assembly
Participants: Sumohit 09:30-21:30
Summary: Colony-PCR with Gibson Assembly of enzyme fragments in pichia vector. GFP investigation in AMP2 samples. PCR with irradiation primers. Media preparation. Gibson Assembly for pBAD18 vector.
Colony-PCR with Gibson Assembly of enzyme fragments in pichia vector
Colony Picking: To expedite the process, 6 colonies of each pichia enzyme fragment were streaked onto a backup plate and incubated at 37°C for 8 hours to obtain sufficient cell material for Colony-PCR. After the 8-hour incubation, cell lysis was performed.
Colony PCR with pichia vector enzyme fragment for new Gibson Assembly
Cells from the streaked colonies were used to perform Colony PCR. The cells were dissolved in 20µl of mM NaOH and heated at 98°C for 45 minutes. Due to insufficient PCR block availability, PCR was not performed and everything was stored at -20°C.
GFP Analysis in AMP2 Samples
After expression, the samples (AMP2-6 and -7) were examined under a fluorescence microscope to check for the presence of GFP. For this, 2 µl of cell suspension from AMP2-6 and -7 samples, as well as 2 µl of saline, were applied to a slide and examined under the microscope.
A GFP-like signal was observed in the AMP2 samples, and there was no signal in the saline Sample. Unfortunately, good images could not be captured, but Caro also observed a GFP-like signal in the AMP2 samples.
Colony PCR with Irradiation Primers
Due to uncertainty about the functionality of Colony PCR using highly redundant primers, it was decided to use some older primers that attach to the Ori. For the test PCR, AMP2-6 was chosen as a positive control for successful cloning, pBAD18 as a positive control, and the samples Mutanase Colony 3, 4, 7, Dispersin B Colony 10, and Dextranase Colony 5. Different PCR runs were performed due to the different sizes of the plasmids. Additionally, a PCR was conducted with Mutanase Colony 3 using AMP primers, suspecting that this colony might represent a successful cloning.
PCR Programs for Various Samples
Program for AMP2-6 and pBAD18:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
66
30
30
Elongation
72
150
30
Final Extension
72
180
Hold
4
for ever
Program for Mut Colony 3:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
66
30
30
Elongation
72
90
30
Final Extension
72
120
Hold
4
for ever
Program for Mutanase and Dispersin B:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
66
30
30
Elongation
72
240
30
Final Extension
72
420
Hold
4
for ever
Program for Dextranase Colony 5:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
66
30
30
Elongation
72
270
30
Final Extension
72
480
Hold
4
for ever
Gel Electrophoresis
Following PCR, the samples were loaded onto a 1% agarose gel and left to run overnight.
LB-Agar medium was prepared for Amp-resistant plates following the protocol.
Transformation of pBAD18-Mut, pBAD18-Disp B, and Empty Vector
Since previously only the DNA constructs from the Gibson Assembly for pichia Gibson were transformed into E. coli cells and plated on the appropriate antibiotic plates, and there were too few LB-Agar plates with Amp, not sufficient for all samples, the Gibson Assembly of enzyme fragments into pBAD18 vector was transformed into E. coli. pBAD18 was used as a positive control for the transformation and plated on Amp-containing plates. Cells without plasmids were treated as a blank during transformation, plated on Amp-containing plates, and used as a negative control for the transformation. The transformation was performed according to the protocol.
Date: Friday, 15.09.2023
Participants: Jessica, Sarah H., Sumohit
Colony PCRs of New Gibson Assemblies with Mut Fwd and His4 New Rev, and Enzym-His4 Sequencing Fwd and M3 (21nt) Rev
PCR Setup for Enzym-His4 Sequencing Fwd and M13 Rev (21nt) Primer
A
Volume [ul]
8 x MM
Q5 2xMM
12.5
100
Enzym-His4 fwd Sequencing-Primer
1.25
10
M13 rev (21nt) Primer
1.25
10
Template
1
-
ddH2O
9
72
PCR Program:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
62
30
30
Elongation
72
115
30
Final Extension
72
300
Hold
4
for ever
PCR Setup for Mut Fwd and His4 New Rev Primer
A
Volume [ul]
8 x MM
Q5 2xMM
12.5
100
Mut fwd Primer
1.25
10
His4 rev neu Primer
1.25
10
Template
1
-
ddH2O
9
72
PCR Program:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
68
30
30
Elongation
72
165
30
Final Extension
72
300
Hold
4
for ever
Linkage PCR: His4 with Dispersin B and His4 with Mutanase
A
Volume [ul]
MM
Q5 2xMM
25
Mut fwd Primer
2.5
His4 rev neu Primer
2.5
Templates (His4 und Mut oder DispB)
Je 3ng
ddH2O
x
PCR Program:
Step
Temperature [°C]
Dauer [sec]
Cycles
Initial Denaturation
98
30
Denaturation
98
8
30
Annealing
67
30
30
Elongation
72
140
30
Final Extension
72
300
Hold
4
for ever
Maintenance
Liquid waste autoclaved
Equipment cleaned
Cell Lysis
The cells in AMP2-6 and AMP1-7 samples were lysed using ultrasound. After lysis, the cells were centrifuged at 14000 rpm for 5 minutes at 4°C, and the supernatant was collected.
Post-Staining Gel for Gibson Plasmids
Since the gel ran overnight without GelRed, in the morning the gel was placed in a GelRed bath with 200 ml GelRed solution containing 60µl Midori, and shaken for 20 minutes. Then GelRed was collected, and the gel was placed in water twice for 30 minutes each, shaking in between.
After cell lysis, the supernatant from AMP2-6 and AMP1-7 samples was examined under a fluorescence microscope to check for the presence of GFP. For this, 2 µl of cell suspension from AMP2-6 and AMP1-7 samples, as well as 2 µl of saline, were applied to a slide and examined under the microscope.
A GFP-like signal was observed in the AMP2 samples, and there was no signal in the saline Sample. Unfortunately, good images could not be captured.
Transformation for Improvement of Existing Part. Cell lysis of pBAD18-Mut/-DspB samples. Preparation of competent cells culture.
Media
LB-Agar medium was prepared for CMR-containing plates.
Prepared as per the protocol.
Transformation of Improvement Plasmids and Empty Vector
As the transformation didn't work well last time and very few cells were visible in the positive control, the procedure was repeated for sample 15D as per the protocol. pBAD18 plasmid was used as the positive control.
Colony PCR of pBAD18-Mut/-DspB Samples
A small amount of cells was taken from the plates streaked on Friday and dissolved in 20 µl 20 mM NaOH. The mixture was incubated for 45 minutes at 98°C. After incubation, the samples were centrifuged, the supernatant was collected, and stored.
Competent Cells Preparation
LB Medium (ÜN) was prepared from BW25113 to create competent cells.
Competent BW25113 cells; Transformation of AMP constructs; Progress of Colony-PCR testing.
Competent Cells
The BW25113 cells were made competent as per the provided protocol and stored at -70°C.
Transformation of AMP Constructs and Empty Vector into Competent BW25113 Cells
AMP1 Colony 7 and AMP2 Colony 6 were transformed into competent BW25113 cells following the provided protocol. pBAD18 plasmid was used as a positive control.
Images
Colony-PCR Progress
Since the previous PCRs didn't work well, new primers that attach to the AOX promoter and AOX terminator were obtained and tested. For the test, Mutanase and Dispersin from the stock were used as DNA templates.
Mutanase and Dispersin B
PCR Program: AOX
PCR Machine: iGEM
Step
Temperature [°C]
Duration [sec]
Cycles
Initial Denaturation
98
30
-
Denaturation
98
8
30
Annealing
66
30
30
Elongation
72
120
30
Final Extension
72
150
-
Preparation of a 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B, Dex1, Dex2, and pichia Vector
Complete 10 µl were loaded from the PCR for Gibson Assembly and 25 µl from the Colony-PCR samples.
Sample Layout
Marker – Mut AOX Test - DspB AOX Test - Marker
Date: Wednesday, 20.09.2023
Experiment: Expression; Colony PCR pBAD18-Mut; Colony-PCR pBAD18-Mut/pBAD18-DspB; PCR for Genomic DNA Integration; PCR Purification and Sequencing; SalI Restriction Digest of His4 Fragments; Overnight Culture; 50x TAE Buffer
Expression of AMP2 and AMP1; Colony-PCR of pBAD18-Mut; PCR for genomic DNA integration; SalI restriction digest of His4 fragments. Overnight culture of Mutanase-Pichia vector colony 1 and pBAD-Dispersin B colony 5 for minipreps. Preparation of 50x TAE buffer.
Expression of AMPs
An expression was performed using AMP2 Colony 6, AMP1 Colony 7, wild-type BW25113 strain, and pSB1C3-GFP-BW25113. One colony was taken and added to 500 ml TB-medium for expression. The cells were incubated at 30°C on a shaker, shaking continuously. During expression, the optical density (OD600) of the samples was continuously measured. Expression was induced by adding 5 ml of 2% arabinose solution at an OD600 of 0.4-0.6. Expression was initiated at 07:00, induced at 13:30, and ended at 21:30.
Colony PCR pBAD18-Mut/pBAD18-DspB
After cell lysis, the cell lysates were used for Colony PCR. The Colony-PCR was performed like a normal PCR for Mutanase and Dispersin B, but using cell lysates as DNA templates and 3´AOX and 5´AOX primers. Samples included Mutanase Colony 1, 5, and 10; Dispersin B Colony 1, 5, and 10. PCR for Mutanase and Dispersin B from the stock was done as a positive control.
Preparation of a 1.0% Agarose Gel for Colony-PCR and Mutanase, Dispersin B
15 µl of the Colony-PCR samples
Sample Layout
Marker – Colony PCR Mut - Mut AOX Control - Marker - Colony PCR DispB - DspB AOX Control
PCR Purification and Sequencing
In the Dispersin B samples, one sample exhibited the correct band. Therefore, the remaining 10 µl of the PCR reaction was purified and sent for sequencing using 3´AOX and 5´AOX primers.
The sample was sent for sequencing using 3´AOX and 5´AOX primers.
PCR for Isolated Genomic DNA from Pichia-Dispersin B Samples
To determine if PCR amplified products other than our desired products, a PCR was done using the isolated genomic DNA from Pichia-Dispersin B samples and 3´AOX and 5´AOX primers. These samples were loaded on the gel along with the Colony-PCR samples.
Colony PCR pBAD18-Mut/pBAD18-DspB
Since no Mutanase sample showed the correct bands, PCR was performed with the remaining 9 Mutanase colony samples as described above.
Preparation of a 1.0% Agarose Gel for Colony-PCR and gDNA
After expression, the samples were examined for GFP in TECAN. Unfortunately, there was no or hardly any difference observed in the fluorescence of AMP2 samples compared to wildtype GFP after normalization.
Above are non-frozen samples and below are frozen samples.
Cell Harvesting
The cells were harvested after expression.
Colony PCR pBAD18-Mut
Since the gel from the previous day did not work well, gel electrophoresis was repeated.
Participants: Sarah (09:30-18:00), Sumohit (09:30-19:00)
Summary:
Restriction digestion of His4 fragments with SalI. Overnight culture of Mutanase-Pichia vector Colony 1 and pBAD-Dispersin B Colony 5 for minipreps. Preparation of 50x TAE buffer.
Expression and GFP Enzyme Kinetics
Since the wrong L-arabinose end concentration was used, and the literature suggests a range of L-arabinose end concentration from 0.01% to 1%, multiple expression attempts were made, and GFP enzyme kinetics were investigated. L-arabinose end concentrations ranging from 0.02% to 2% were used for expression and GFP analysis.
Remaining Sequencing Reactions at Microsynth
Clean-up of His4 Fragments
Performed with a kit from NEB. Elution in 8 ul.
Sample
Conc. [ng/ul]
260/280
260/230
His4 1
51.4
1.93
1.73
His4 2
60.7
1.87
1.61
Disposal of n-Hexane and possibly tert-Butanol
Fill into a container, store, and fill out the form together with Martina.
Restriction Digestion of Minipreps with SalI-HF for Pichia Transformation
Component
Volume [ul]
Comments
Minipreps (approx. 300ng)
Disp B Col5: 6 ul; Mut Col1: 12 ul
Cut Smart
1.5
SalI-HF
0.5
ddH2O
Disp B Col5: 7 ul; Mut Col1: 1
Digest for 15 minutes at 37°C, then heat inactivate for 20 minutes at 65°C. No gel!
Transformation of Digested Plasmids into Pichia pastoris
Transformation was carried out as described in the Micro scale protocol of the Frozen-EZ yeast Kit, with the deviation that 20 ul of competent cells were used instead of 10 ul.
Date: Monday, 25.09.2023
Experiment Update: Pichia Transformation Results and Overnight Culture
Checked Pichia transformation plates - no colonies but filamentous fungal growth observed.
Overnight Culture of pBAD Mut3
5 ml LB+Amp with 0.5% (250 mg) arabinose inoculated with 50 ul overnight culture. Sent to Herzberg for TECAN measurement.
Date: Tuesday, 26.09.2023
Experiment Update: Gibson Assembly and Expression Troubleshooting
Participants: Sarah; Sumohit (09:30-19:00)
Gibson Assembly of Dispersin B and Dextranase in pBAD18 Vector
Since there was a suspicion that the Dispersin B cloning was not successful (there was a suspicion that His4 was not cloned properly), the Dispersin B cloning was repeated from the PCR step. An attempt was made to clone dextranase into pBAD18. On Tuesday, only the PCR and gel electrophoresis were performed.
Sample
Amount for Gibson [ng]
Volume of Sample [µl]
Water [µl]
NEBuilder [µl]
Mut
110.5
1.44
6.67
10
Dsp B
101.9
1.31
6.8
10
Dex 1
Dex 2
His4
98.2
1.09
pBAD18 Vector
75
0.80
Gel Electrophoresis:
Marker - Disp B - Dex 1 - Dex 2 - Marker
Expression Troubleshooting
Since the expression did not work, a setup was prepared with AMP2-6 in BW25113 and NEB stable cells. The same cells were seeded in wildtype and with pSB1C3-GFP in TB medium at OD 0.4 with various L-arabinose concentrations ranging from 0.02% to 2%. Enzyme kinetics were measured in a reader after induction. 5 ml setups were also prepared so that if successful, the cells can be lysed and analyzed on SDS-PAGE. Unfortunately, after 4 hours of induction, no increase in fluorescence was observed in any of the samples.
3x 5ml Overnight Cultures for pBAD-Dispersin B Kol5
Date: Wednesday, 27.09.2023
Experiment Update: Cell Lysis and Minipreps
Cell Lysis of Various Samples by Sumo
Cells were pelleted at 5000g and RT for 5 minutes, resuspended in 4 ml 0.9% saline, and subjected to ultrasound pulses on ice for 30 seconds, repeated 5 times. A 1-minute break was taken between cycles to prevent overheating. The cell extracts were transferred to Eppendorf tubes and centrifuged at 14000 rpm and 4°C for 60 minutes. The pellet and supernatant were separated.
Gel Electrophoresis:
Marker - Disp B - Dex 1 - Dex 2 - Marker
Minipreps of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3
Performed using the Analytik Jena kit, where 3 overnight cultures of Dispersin B were loaded onto a column. Eluted in 60 µl.
Sample
Concentration [ng/ul]
260/280
260/230
pBAD-Dispersin B Kol5
117.7
1.96
2.05
pBAD-Mutanase Kol3
76.3
1.94
2.06
Preparation of LB Medium
Prepared 500ml of LB medium following the standard recipe.
Summary: Gibson Assembly of Dispersin B and Dextranase in pBAD18 vector. Expression troubleshooting with various concentrations of L-arabinose. Bradford assay for the previous expression. Sequencing of Gibson samples. Restriction digestion of Gibson samples. DNA cleanup of the restriction-digested Gibson samples.
Gibson Assembly of Dispersin B and Dextranase in pBAD18 Vector
After observing bands on the gel, 10 µl of the PCR samples were purified and further processed with Gibson Assembly.
Sample
Concentration [ng/µl]
A260/A280
A260/A230
Dispersin B
33
1.79
2.41
Dex 1
28.5
1.72
1.99
Dex 2
28.8
1.84
2.53
His4
34.6
1.82
1.93
Sample
Gibson Amount [ng]
Sample Volume [µl]
Water
NEBuilder
Dispersin B
110.5
3.35
3.01
10
Dex 1
#VALUE!
#VALUE!
10
Dex 2
#VALUE!
His4
98.2
2.838150289
pBAD18 Vector
75
0.80
Transformation of Gibson Constructs for pBAD18 Gibson
Gibson Assembly for pBAD18 Gibson constructs was transformed into E. coli cells and plated on respective antibiotic-containing plates. pBAD18 was used as a positive control for transformation and plated on amp-containing plates. Cells without plasmids were treated as blank during transformation, plated on amp-containing plates, and serve as the negative control for transformation. The transformation was carried out as per the protocol.
Expression Test from Tuesday - Bradford Assay
To be able to do SDS-PAGE later, protein concentration was measured from the expression samples. The protein concentration was measured as in the protocol.
BW25113
BW-1
BW-2
BW-3
BW-4
BW-pSB1c3
Dilution
1
1
1
1
1
1
Protein [µg/ml]
28.7578863
19.6601932
21.6451444
15.6489375
15.4835249
18.5023049
On SDS Page
7.5
7.5
7.5
7.5
7.5
7.5
Amount on SDS
215.684147
147.451449
162.338583
117.367032
116.126437
138.767287
BW25113
N-1
N-2
N-3
N-4
N-pSB1C3
Dilution
1
1
1
1
1
1
Protein [µg/ml]
24.2090397
5.84824082
13.8707521
10.3557342
10.3143811
17.427123
On SDS Page
7.5
7.5
7.5
7.5
7.5
7.5
Amount on SDS
181.567798
43.8618061
104.03064
77.6680068
77.3578582
130.703423
B Stands for BW25113; N for NEB Stable; 1 for 0.02% L-Arabinose; 2 for 0.5% L-Arabinose; 3 for 1% L-Arabinose; 4 for 2% L-Arabinose.
No SDS-PAGE was conducted because no differences in fluorescence were observed among all samples.
Expression Test 2
Since the previous expressions did not work, a test was conducted. BW25113 cells with AMP2-6 and NEB stable cells with AMP2-6 were cultured in LB medium. The cells were induced with varying L-Arabinose concentrations from 0.02% to 0.2% L-Arabinose. After induction, the cells were incubated overnight at 37°C on a shaker.
Sequencing pBAD-Mutanase Kol3 and pBAD-Dispersin B Kol5
SalI HF Digestion of pBAD-Dispersin Kol5 and pBAD-Mutanase Kol3 for Transformation
1.5 µg Plasmid/ Digestion
Component
Volume [µl]
Comments
Minipreps (Disp: 1.5 µg and Mut: approx. 1.3 µg)
Disp: 12.7 ; Mut: 17
Cut Smart
5
SaliHF
1
ddH2O
Disp: 31.3 ; Mut: 27
15 min digestion at 37°C and then 20 min heat inactivation at 65°C.
PCR Cleanup of Restriction Digests
PCR cleanup of the restriction digests was unsuccessful because Zymo was used instead of NEB, and Zymo requires a larger elution volume. Therefore, two overnight cultures (ÜN Kulturen) of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3 were prepared.
After expression, OD600 and fluorescence of the expression samples were measured.
Sample
BW
1b
2b
3b
4b
5b
1n
2n
3n
4n
5n
b-p
n-p
Fluo
413.00
525
477.000
514.000
643.000
547.000
559.000
502.000
504.000
641.000
590.000
10071
6937.000
Fluo
384.00
485
498.000
493.000
661.000
490.000
512.000
503.000
502.000
617.000
561.000
10034
7148.000
Fluo
410.00
503
486.000
528.000
698.000
505.000
512.000
499.000
499.000
695.000
536.000
9940.000
7234.000
MW Fluo
402.33
504.33
487.00
511.67
667.33
514.00
527.67
501.33
501.67
651.00
562.33
10015.00
7106.33
OD600
5.89
2.90
2.68
2.69
3.82
2.84
1.97
2.06
1.86
2.93
4.93
5.42
4.13
Fluo/OD
68.31
173.91
181.72
190.21
174.69
180.99
267.85
243.37
269.71
222.18
114.06
1847.79
1720.66
Fold Change
1.00
2.55
2.66
2.78
2.56
2.65
3.92
3.56
3.95
3.25
1.67
27.05
25.19
For pSB1C3-GFP, a strong GFP signal was seen in both BW25113 and NEB stable approach compared to wild type samples. In the other samples, only a slightly increased GFP signal was seen compared to the BW25113 wild type.
Cell Harvesting
After the cells, especially the pSB1C3-GFP samples, showed GFP, the cells were lysed. The cell lysis was performed as per the protocol.
Bradford
To perform SDS-PAGE later, the protein concentration of the expression samples was measured.
Sample
BW
1b
2b
3b
4b
5b
1n
2n
3n
4n
5n
b-p
n-p
Fluo
413.00
525
477.000
514.000
643.000
547.000
559.000
502.000
504.000
641.000
590.000
10071
6937.000
Fluo
384.00
485
498.000
493.000
661.000
490.000
512.000
503.000
502.000
617.000
561.000
10034
7148.000
Fluo
410.00
503
486.000
528.000
698.000
505.000
512.000
499.000
499.000
695.000
536.000
9940.000
7234.000
MW Fluo
402.33
504.33
487.00
511.67
667.33
514.00
527.67
501.33
501.67
651.00
562.33
10015.00
7106.33
OD600
5.89
2.90
2.68
2.69
3.82
2.84
1.97
2.06
1.86
2.93
4.93
5.42
4.13
Fluo/OD
68.31
173.91
181.72
190.21
174.69
180.99
267.85
243.37
269.71
222.18
114.06
1847.79
1720.66
Fold Change
1.00
2.55
2.66
2.78
2.56
2.65
3.92
3.56
3.95
3.25
1.67
27.05
25.19
BW stands for BW25113;NEB stands fpr NEB stable cells, 4 stands for 0.15% L-arabinose induction: 5 stands for 0.2% L-arabinose induction:
Expression Troubleshooting 3
Since this expression provided better results, a new setup was arranged without aluminum foils as it showed better results in Expression Troubleshooting 2 with these measures. Overnight cultures were set up, inoculated, and allowed to grow at room temperature over the weekend for expression.
Minipreps of pBAD-Dispersin B Kol5 and pBAD-Mutanase Kol3
Sharkey
Restriction Digest for Transformation in Pichia pastoris
Component
Volume [ul]
Comments
Minipreps (Disp: 1.5 µg and Mut: approx. 1.5 µg)
Disp: 12.7
Mut: 14.4
Cut Smart
5
SaliHF
1
ddH2O
Disp: 31.3
Mut: 29.6
15 minutes at 37°C followed by 20 minutes heat inactivation at 65°C.
PCR Cleanup of a pBAD-Dispersin B Restriction Digest
Performed with NEB kit. Eluted in 6 µl.
Sample
Conc. [ng/ul]
260/280
260/230
pBAD-Dispersin B Kol5
Approx. 80 ng/ul? (as written on Eppi)
?
?
Gel after Restriction Digest
0.7% Gel
Expected bands: Disp: approx. 9362 bp and Mut: approx. 9551 bp
150 ng per lane
Not cleaned-up Restriction Digest: 5 µl each
Transformation in Pichia pastoris
For the transformation in Pichia pastoris, the EZ-Kit protocol was followed with the following adjustments:
Transformation Setup Details
For pBAD-Dispersin B Kol5 (without clean-up):
30 ul competent cells + 25 ul digested DNA
For pBAD-Dispersin B Kol5 (with clean-up) and pBAD-Mutanase Kol3:
This experiment involved activities related to medium preparation, expression, harvesting, fluorescence measurement, sequencing, and maintenance.
Medium Preparation
Since the available media appeared turbid, as if something had grown in them, 1 L of LB medium was prepared and autoclaved.
Expression
After autoclaving and cooling the medium, the expression process began. 200 ml of LB medium with the appropriate antibiotics were added to a flask, followed by the addition of 2.22 ml of the preculture to the main culture. Expressions were performed using AMP2 colony 6, AMP1 colony 7, wild-type BW25113 strain, BW25113 with pBAD18 empty vector, and pSB1C3-GFP-BW25113. The cells were incubated at 37°C on a shaker with continuous shaking. The optical density (OD600) of the samples was continuously measured during the expression. When the OD600 reached 0.7-0.75, the expression was induced by adding 4 ml of 10% arabinose solution. The expression was started at 10:00 AM, induced at 12:30 PM, and concluded at 9:30 PM.
Harvesting
After the expression, the cells were harvested following the protocol.
E. coli NightSeq Sequencing of Gibson Colonies
For sequencing, 10 colonies from both Dex and Disp plates were picked using Enzyme-His4 Sequencing Primer and 5' AOX Primer in separate tubes. Microsynth tubes were labeled and spun down, and colonies were picked and resuspended in the Microsynth tube. Subsequently, they were streaked on an LB-Amp plate using toothpicks. Additionally, 100 ul of 10uM primer solutions were prepared in separate tubes.
Note: Sequencing quality was not satisfactory, likely due to the colonies being too small.
Date: Tuesday, 03.10.2023
Date: Wednesday, 04.10.2023
Participants:Sarah H.
Cell Lysis of Various E. coli Cell Samples
Samples 26, 26R, 17, and 17R were resuspended in 6 ml of 0.9% saline. Samples B, P, and TB were transferred to Falcon tubes. The cells were lysed using ultrasound with 5 pulses of 30 seconds each and 1-minute intervals between the pulses. The solutions were centrifuged at 13,000 rpm for 60 minutes at 4°C. The supernatant was collected and frozen along with the pellets.
OD Measurement of Shake Flask Cultures and Pelleting of Samples
Kulture
OD(600) 1:50 Dilution
OD(600) Undiluted
Dilution for OD=6
30 ml Approach (Culture + ddH2O)
AMP 1-7
0.142
7.1
1:1.18
25.4 + 4.6
AMP 2-6
0.117
5.85
-
-
AMP 1-7 R
0.151
7.55
1:1.26
23.8 + 6.2
AMP 2-6 R
0.158
7.9
1:1.31
22.9 + 7.1
Gr.1 ?
0.085
4.25
-
-
ASB ?
0.178
8.9
1:1.48
20.3 + 9.7
Gr.6?
0.176
8.8
1:1.46
20.5 + 9.5
Pelleting of Cell Samples: Centrifuged at 5000g for 15 minutes.
Date: Thursday, 05.10.2023
Participants: Jana, Sarah H., Sumohit
Cell Lysis of Various E. coli Cell Samples
Cell samples were resuspended in 6ml of 0.9% saline. The samples were transferred to Falcon tubes. The cells were lysed using ultrasound with 5 pulses of 30 seconds each and 1-minute intervals between the pulses. The solutions were centrifuged at 13,000 rpm for 60 minutes at 4°C. The supernatant was collected and frozen along with the pellets.
Bradford Assay for Protein Concentration Determination
Preparation of the Calibration Curve:
BSA Amount [ug]
BSA Stock [ul]
H2O [ul]
0
0
100
2
2
98
5
5
95
7
7
93
10
10
90
15
15
85
20
20
80
900 ul of Bradford reagent added, mixed, and incubated for 5 minutes. Measurement at 595 nm in the spectrophotometer.
Measurement of Samples:
Approach in cuvettes:
100ul of a 1:10 dilution of the samples
900ul Bradford reagent
Mix and incubate for 5 minutes. Measurement at 595 nm in the spectrophotometer.
Sample
B
C
D
E
Dilution
1
1
1
1
Protein [ug/ml]
On SDS PAGE (7.5%)
Quantity on SDS
Date: Friday, 06.10.2023
Participants: Jana, Sarah H., Sumohit
Sample Preparation for SDS PAGE
A total of 15ul of samples were prepared as follows:
7.5 ul of sample
7.5 ul of 2x loading buffer
Heated at 98°C for 10 minutes. Samples were frozen for future use.
Date: Monday, 02.10.2023
Participants: Jana, Sarah H.
Sample Preparation of Pellets for SDS PAGE
For each sample:
- 150 / OD = Volume of the respective cell sample
- Centrifugation: 14000g, 4°C, 20 min
- Discard the supernatant
- Resuspend the pellet in 7.5 ul of water
- Add 7.5 ul of 2x SDS loading buffer
- Heat at 98°C for 10 minutes
- Samples were frozen until further use (-20°C)
Date: Tuesday, 03.10.2023
Participants:Jana, Sumohit
Procedure:
Arrangement:
(Top: Gel1, Bottom: Gel2)
Apply 120V for 1 hour.
Stain the gel in Coomassie for 30 minutes and destain for 1 hour.
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