Notebook

• Prepared three sets of agar plates with antimicrobials as a selection mechanism, with concentration 50 mg /mL (kanamycin, chloramphenicol), for a total of nine plates
• Carried out a heat shock transformation following this protocol

  Heat Shock Transformation Protocol

  1. Thaw competent cells on ice
  2. Dilute 220 ng /µL mCherry plasmid down to 1ng /µL by taking 4.5 µL and making up to 1 mL stock

• Transformations from 26/6/23 had failed. No colonies were visible.
• Prepared more agar plates, with kanamycin resistance (50 µg /mL). Spread different concentrations of cells over these (1:1, 1:10, 1:100) per set of plates, from liquid culture
• Competent cells were from: a. Our own batch, b. Batch donated by Brooks, and c. from Easter outreach session
• This is to determine if it is our competent cells that are faulty
• Carried out a heat shock transformation to protocol

• The transformations from 27/6/23 succeeded, and colonies were visible:
• Highest number of colonies on all the 1:1 plates, with many more coloies on ours and Brooks' batches of competent cells, compared to the outreach batch
• Brooks' an our batch of competent cells had roughly the same DH5α division rate, both are higher than the outreach batch.
• Colonies from transformed outreach and our batches were grown in an overnight culture, which should allow us to practise mini-prepping and electrophoresis tomorrow. This will also allow us to confirm the presence of the mCherry plasmid in our cells
• Average competent cell efficiency found to be 146 545 CFU /µg

Cell concentration	Number of colonies present	CFU /µg
1:1	13	174 909
1:10	N/A	N/A
1:100	1 924	118 182

• 1:10 plates had very few colonies present, especially when compared to 1:1 and 1:100, so we assume something went wrong. Have not included it in efficiency calculations.
• Overnight cultures of our transformed competent cells were made, with transformed outreach cells left to grow
• iGEM registry contradicts the Airtable for the antibiotic resistance in the Anderson promoter plasmid backbones (ampicillin vs chloramphenicol), so we performed an alignment in Benchling to determine resistance
• Our Anderson promoters are resistant to chloramphenicol

• Mini-prepped 6 overnight cultures, with 3 repeats of each culture (using protocol in Nippon mini-prep kit)

• Attempted to find the entire sequence of the mCherry plasmid we had used to confirm our competent cells were indeed competent
• We were only able to find the sequence of the gene

• Ran restriction digests of mCherry with BsmbI
• 'Bio Bois' WhatsApp group provided us with the plasmid backbone of mCherry (pET28b)
• BsmbI restriction enzyme has three restriction sites on the plasmid backbone
• Due to uncertainty in the assembly of the construct, the BsmbI restriction sites may have been removed
• Carried out restriction digests with DNA from our outreach session at Easter, and DNA we had assembled. Followed the time-saver protocol

Lane	Contents
1
2
3	Ladder
4
5	AN Outreach (TB)
6
7	AN Self Made (AN)
8
9	M6 Self Made (Mo)
10
11	M6 Outreach (Mo)
12
13	BST Outreach (max)
14
15	Negative Control (growth slower)

• Results of the restriction digest:
• The last 2 lanes were unexpected, as the last lane (15) is a negative control, and lane 13 is outreach DNA. There may have been confusion with the labelling of lanes, or samples being mixed up, however

• Prepared PCR mixes for the four growth slowers we are characterising (gp2, AsiA, gp240, gp0.7), following this protocol
• The growth slower template DNA and primers were from gBlocks, from the 2022 iGEM distribution kit
• Primers were resuspended to 100 µM concentration, then diluted to 10 µM concentration for the PCR, following this protocol

• Using the three positive results from our electrophoresis, gel extraction was used to excise the DNA, which was then frozen
• The resitriction digest of AsiA (growth slower) failed, as a bubble was present in the gel
• Prepared PCR mixes for the growth slower genes (gp104, gp79, Alc, AsiA) that had yet to be PCRd
• Prepared another 1% agarose gel for running the PCR products and mCherry plasmid
• The PCR product was loaded to the agarose gel with 5 µL of loading dye
• The mCherry plasmid from amplification in competent cells was loaded to the gel aslo (10 µL DNA, 2 µL loading dye)

• Transformed five Anderson promoters, ribosome binding sits, and terminators (all from the iGEM distribution kit), following the iGEM transformation protocol
• Transformed the mCherry plasmid as a control
• Prepared agar plates with 50 µg /mL chloramphenicol
• Transformed cells were then incubated on chloramphenicol agar plates, and incubated overnight at 37 C

• Of the plates with the transformed cells growing from yesterday, only the mCherry plate had colonies present
• The transformation was redone, with 4 µL DNA used to make the competent cells, as well as using different competent cells that were expected to give more reliable results. This was done following the iGEM transformation protocol. It is expected that a higher concentration of DNA, and lower chloramphenicol concentration, should result in greater chances of success with these transformations
• Made new chloramphenicol agar plates, at 25 µg /mL concentration, for the transformations above

• Attempted to retransform the ribosome binding site (RBS) (BBa_0034) and the consensus Anderson promoter (BBa_J23119)
• Things thought to have gone wrong
• The promoter DNA had no red dye in
• This could be rectified by spinning the plate, due to manufacturing error, or the pellet may have been lost somewhere
• Therefore:
• Remaining J23119 DNA was pooled from the 2023 iGEM distribution kit (which had no colour), with the same DNA from the 2022 distribution kit (which had a slight orange colour)
• 10 µL water added to wash the residual DNA from the 2022 plate, and then transformed 33 µL NEB DH5α cells with 10 µL of the resuspended DNA (with the remainder moved back to the tube containing the resuspended 2023 distribution kit DNA)
• Problem no. 2:
• The DNA labelled as the RBS was not the RBS
• The tube of resuspended DNA was labelled ambiguously (1(I14) or 1(I19))
• Checked the piercing of foil on the distribution plate, and I14 was pierced
• I14 contains a JUMP plasmid with no resistance to chloramphenicol, explaining the failure for transformant colonies to grow
• Set up new transformations, with the DNA from the correct well (1(I19))
• These transformations were found to be successful

• Plated results of the transformation on LB agar, 5 µL of 50 mg /mL chloramphenicol into 50 mL agar
• Results of the transformation:

Promoter	Number of Colonies
J23100	0
J23106	30
J23110	40
J23114	80
J23119	0
B0015	0
B0019	0

• Repeated the negative results (which produced 0 colonies) with 1 µL DNA to 50 µL competent cells
• 30 mins on ice, then heat shock at 42 C for 45s
• BBa_B0034 was not transformed, as we had been provided with it pre-transformed
• Brooks brought RBS (BBa_0034) and consensus Anderson promoters (BBa_J23119) plates with colonies
• Prepared overnight cultures of J23106, J23110, J23114 for miniprep DNA extraction. 1 positive colony from each plate was grown in 10 mL liquid culture (LB broth), with 25 µg /mL chloramphenicol

• No colonies were present on BBa_B0015, or BBa_J23100 after overnight incubation at 37 C. These dishes were placed back in the incubator in the hopes of further growth
• Minipreps for BBa_J23119, BBa_J23106, BBa_J23114, BBa_J23110, and BBa_B0034 (RBS) were completed (as per protocol)
• Attempted to retransform BBa_B0015 and BBa_J23100 again, using competent cells from iGEM Sheffield's PI, Dr. Andy Hitchcock. These transformations used up the last of the DNA samples in the 2023 iGEM distribution kit

• Resuspended three terminators from the 2023 iGEM distribution kit, which were then transformed into into competent cells
• Transformed LB-agar plates from 11/7/23 that had successful growth after incubation for 48 hours. The identity of these bacteria needs confirming to ensure they are as expected
• Grew the transformed cells on LB-agar plates, with two plates per transformation. One of these plates was old, and the other new

Terminator	Old plate	New plate
B0012	Failed	Succeeded
rnpB_T1	Failed	Succeeded
rpoC_T	Failed	Succeeded

• Made liquid cultures to confirm the antibiotic resistance of colonies from BBa_J23100, BBa_J23119, and BBa_B0015
• These were made from a single colony isolated from a plate. Grown in 5 mL LB broth liquid cultures with 2.5 µL of 50 mg /mL chloramphenicol solution
• These liquid cultures were incubated at 37 C, 250 RPM for one hour
• Nanodropped cultures of BBa_J23110, BBa_J23119, BBa_J23114, and BBa_B0034, which were miniprepped on 11/7/23

	Concentration ng / µL	260/230	260/280
RBS	21.812	1.71	3.2
J23106	23.614	1.71	3.45
J23110	50.382	2.39	2.36
J23114	6.660	1.15	3.71
J23119	21.725	1.87	3.26

• BBa_J23114 is to be repeated, due to the low concentration

• BBa_J23119, BBa_J23100, and BBa_B0015 liquid cultures are clear, so it is likely the plates were contaminated. These liquid cultures will be remade using alternative plates

J23119	Making two liquid cultures, using plates from Brooks (backup and normal)
J23100	Using alternative plate made on 6/7, stored in fridge Also being repeated by Brooks
B0015	Using alternative plate made on 6/7, stored in fridge Brooks is also repeating
Other terminators	Making liquid cultures of B0012, rnpB-T1, rpoc-T, for if B0015 does not work again

• Checked overnight samples that had visibly gone cloudy, measuring optical density at 600 nm in 1 mL cuvettes with an LB blank

	OD600
B0034	0.916
J23114	0.924
J23110	1.012
J23106	1.076

Absorbance of successful overnight cultures

• Successful overnight cultures may need stepping up as the ODs are >0.9 ABS, to allow the bacteria to enter log phase for longer term storage
• Stored BBa_J23114, BBa_J23106, BBa_J23110, and BBa_B0034 at -70 C in 50% glycerol, made from the high OD stocks, without the step up of cultures

Restriction Digest Predictions

• Using the sequence in the Airtable for the 2023 iGEM distribution kit, 'digital restriction digests' were run to predict the outcomes of the restriction digests. This allowed the verification that the products were correct

Part	Restriction enzyme	Cut sites	All fragment lengths	Fragment length we need
B0034	BsaI	2204, 2230	26, 2385	26
B0015	BsaI	2204, 2339	135, 2385	135
J23100	BsaI	2204, 2243	39, 2385	39
J23106	BsaI	2247, 2286	39, 2428	39
J23110	BsaI	2242, 2203	39, 2385	39
J23114	BsaI	2204, 2243	39, 2385	39
J23119	BsaI	2204, 2203	39, 2385	39
J23104	BsaI	2204, 2203	39, 2385	39

Mini-Preps

• Mini-preps were completed for as many liquid cultures as possible, reducing the wastage of liquid culture where possible
• Mini-preps of BBa_J23106, BBa_J23110, BBa_B0034 (RBS), BBa_J23119, and BBa_J23114 were completed with old-source liquid cultures stored at 4 C, except BBa_J23114, as the liquid culture for this failed and a new one was made with the same protocol
• Should it not be possible to obtain liquid culture of BBa_J23100, the next strongest Anderson promoter (BBa_J23102) will be used. This Anderson promoter is also found in the 2023 iGEM distribution kit
• Brooks advised a redo of the liquid culture of BBa_J23119, rpoc-T, rnpB_T1, BBa_B0015, and BBa_B0012, without storing at 4 C between culturing overnight and using the liquid cultures
• Redos of the liquid cultures of BBa_J23100, BBa_B0015, BBa_B0012, rnpB_T1, rpocC_T, and BBa_J23114, as some of the liquid cultures did not work as expected
• The miniprep concentrations of these components will also be confirmed, allowing the determination of whether new cultures are required for these

• Carried out PCR on all growth slowers, using DNA stock of 1 ng/µL
• Restriction digests of promoters and RBS with BsaI-HF, to ensure all parts are working correctly. Quantities shown in table below
• Also ran a gel of growth slowers, with promoters: BBa_J23100, BBa_J23119, BBa_J23104 and RBS

• PCR reaction mix table:

Part	DNA (µL)	NE buffer (µL)	BsaI (µL)	H2O (µL)
J23114	10.2	5	1	33.8
J23100	9.9	5	1	34.1
rpoC-T	7.9	5	1	36.1
rnpB_T1	11.8	5	1	32.2
B0015	18.3	5	1	25.7
B0012	10.9	5	1	33.1

• RESULTS

Growth Slower	Status
J23100	Successful
gp2	Successful
J23119	Successful
RBS (B0015)	Successful
gp240	Successful
gp0.7	Successful
gp79	Successful
AsiA	Successful
Alc	Successful
J23104	Unsuccessful
gp106	Unsuccessful

• Successful bands were excised and placed in tubes for digestion
• Unfortunately, no photograph taken of gel as the lab was closing therefore we decided to repat this gel to have more complete qualitative results

• Carried out gel extraction protocol of the bands excised on 17/7/23
• Minipreps of B0015, rnpB_T1, rpoC_T1, J23100, J23114. Miniprep Protocol
• Glycerol stocks made (Creating a Glycerol Stock)

• Prepared a gel ( video transcript from gel preparation), as well as overnight cultures of BBa_B0015, BBa_J23100, BBa_J23114 (5 mL LB broth, 1 µL of relevant antibiotic, swabbed colony from an agar plate into the broth, and incubated overnight at 37 C, 200 rpm).

• Carried out minipreps of J23100, J23114, B0015 overnight cultures which were then nanodropped measure how much DNA was in them. Miniprep Protocol

Part	Concentration, ng /µL	260/230	260/280
J23100	55.869	1.48	1.78
J23114	55.012, after concetrating 15.281	1.84	1.76
B0015	58.474	1.83	1.83

• J23114 was concentrated by spinning in a vacuum centrifuge, due to the DNA being in an elution buffer, and thus not able to be nanodropped

• Carried out restriction digests of minipreps competed on 20/7

Part	DNA (µL)= 1µg	NE Buffer (µL)	Bsal (µL)	H2O(µL)
J23114	18.18	5.0	1.0	26.0
J23100	17.9	5.0	1.0	26.0
B0015	17.1	5.0	1.0	27.0

• Added 20 µL to BBa_J23114 stock inadvertently, halving concentration (~27.9 ng /µL)
• PCR products will be purified from the gel on Monday 24/7 (gp0.7, gp2, gp104), along with the gel made today (gp240, gp79, AsiA)
• Alc has not shown up as expected in either gel electrophoresis

• Transformed competent cells with BBa_J23114 (from the 2022 iGEM distribution kit), with efasGFP and pJUMP28-1A (both from the 2023 iGEM distribution kit), poured onto agar plates and incubated at 37 C overnight.
• Redid the PCR of Alc, and purified. PCR Purification
• Ordered vsfGFP-0, Pseudomonas butanovora 1-butanol dehydrogenase BOH precursor gene

• pJUMP28-1A and efasGFP plates are all successful
• J23114 unsuccessful, as there was no growth on the agar plates from the overnight culture
• Checked all concentrations with the nanodrop for pure DNA parts, and recorded in spreadsheet
• Overnight cultures setup in 5 mL LB broth for pJUMP28-1A and efasGFP transformations
• No all minipreps were completed with the same kit, so we will ensure the correct elution buffers are used as a blank, to ensure the curves have 'normal behaviour'

• Miniprepped pJUMP28-1A and efasGFP to this protocol
• Determined concentrations of pJUMP28-1A, efasGFP and Alc which we found in an already purified, from touchdown PCR on 24/7/23, in an already miniprepped and purified form. We don't really know when this Alc was done. Made a plan to be more on top of progress of our genes. )
• Set up an overnight culture of BBa_J23119 in 5 mL LB broth, from a plate stored in fridge
• Digest of the table below with protocol. Protocol

Part	DNA, µL	10× rCutSmart Buffer, µL	BsaI, µL	H2O, µL
J23114(T2)	1.02	1	0.5	7.482
J23100(T2)	3.45	1	0.5	5.049
J23106(T1)	1.81	1	0.5	6.689
B0034 (RBS)(T1)	1.66	1	0.5	6.84
B0015(T1)	1.72	1	0.5	6.78
J23110(T1)	1.87	1	0.5	6.628
pJUMP28-1A	0.47	1	0.5	8.033
efasGFP	3.3	1	0.5	5.199

• Researched the gene of the biosensor being used (BmoR):
• We know that there is a transcription factor-promoter pair (BmoR-P_BMO) in the gene, of which we know the whole gene sequence and the sequence that codes for the biosensor producing gene (transcription factor-BmoR). But we don't know which the sequence that corresponds to the inducible promoter that responds to that transcription factor (\(\text{P}_{\text{BMO}}\)) is. We will find it.
• In theory, the biosensor gene produces a transcription factor always; that binds to a small molecule produced by the gene of interest, and this complex binds to an inducible promoter that initiates translation of the growth modulator/reporter gene.
• Ran colony PCRs from plates that had been colonised by E. Coli DH5α transformed with the assembly plasmids based on the Protocol on the iGEM website.Protocol here.
• Ran PCR products (some with NcoI) on 3% agarose gel

Lanes	Contents
1
2	Ladder (1kb)
3	J23100
4	J23106
5	J23110
6	J23114
7	B0034 (RBS)
8	B0015 (terminator)
9	efasGFP

• Assemblies transformed into DH5α
• LB agar 2.5% plates with kanamycin (50 mg /mL) used

• Carried out Colony PCR (Protcol TBC)
• Per PCR:
• 20 µL master mix
• 1.6 µL each primer(fwd, rev)
  PS1:AGGGCGGCGGATTTGTCC; PS2:GCGGCAACCGAGCGTTC
• 400 µL Taq polymerase
• 396.8 µL sterile distilled water
• Run PCR product in a 3% agarose gel for 180m at 100V, with 1 µL of loading dye per 5 µL DNA
• Excise expected sections from gel, produce overnight cultures
• Colony PCR primers resuspended by adding distilled sterile water and left in original tubes in a 100 mM solution
• Colony PCRs left overnight in the thermocycler on hold at 4 C
• Kanamycin plates made on Friday (28/7/23) may have 1/10 the kanamycin they should have had. May explain the mixture of fluorescent and non-fluorescent colonies present on contaminated plates

• Digested BBa_J23119 with BsaI-HFv2, and ran on a gel. This was then used to make an assembly with it
• Ran colony PCRs on a 3% gel at 100V for 2 hours
• Analysed and chose good colonies (potentially using NcoI restriction sites and running on a gel again), and then produced liquid cultures

• More competent cells were also prepared, as per this protocol
• Transformed pJUMP29-1A, following the the iGEM protocol

• Restriction digested all of the miniprepped colony PCRs with BsmbI and NcoI, to avoid circular plasmids. Made predictions of the restriction digest in Benchling:

• The BBa_J23100 promoter may need to be used, however it doesn't always work, to allow the gel to complete. This explains with the constructions in the table below do not show on the gel from 01/08/2023

Colonies to digest
J23100	2, 3, 5, 7
J23110	2, 4, 7, 8
J23106	1, 5, 6, 7 (due to it being unexpected), 9
J23114	2, 3, 5, 7, 9

• Digested both pJUMP28-1A and pJUMP29-1A with BsaI, and then assembled in to the backbone of pJUMP28-1A with the insert from pJUMP29-1A

• Mini-prepped BBa_J23104, and then ran on a gel
• Ran BBa_J23119 digest on a gel
• Miniprepped pJUMP29-1A, then ran on a gel, and used the Nanodrop to determine the concentration of DNA
• Also miniprepped colony PCR cultures, then used the Nanodrop to determine the concentration, and digested the product with BsmbI and NcoI
• Also digested pJUMP28-1A and pJUMP29-1A, which were also Nanodropped, and then assembled together in a thermocycled

• 1 µg of DNA used for the BsmbI and NcoI digests, shown in the table below:

Construct	Concentration (ng /µL)	Volume (µL)
J23100, 2	66.020	15.1469… (15.1)
J23100, 3	35.177	28.4276… (24.4)
J23100, 5	52.452	19.0650… (19.1)
J23100, 7	62.581	15.9792
J23110, 2	29.524	33.8707
J23110, 4	91.204	10.9644
J23110, 7	196.934	5.0778
J23110, 8	32.679	30.6006
J23106, 1	255.645	3.9116
J23106, 5	67.05	15.6128
J23106, 6	18.084	55.2975
J23106, 7 J23106, 9  270.744 J23114, 2  180.796 J23114, 3  136.294 J23114 ,5  205.870 (260/230!) J23114 ,7  219.210 J23114 ,9	154.714	6.4635

• Made more digests of pJUMP28-1A and pJUMP29-1A

Components	Volume	Vol for pjump28	Vol for pjump29
DNA	1ug	4.67ul
10x NEBuffer		5ul	5ul
BsaI		1ul	1ul
H2O		39.33ul	20.75ul

• However, we do not have enough pJUMP29-1A to make the second construct
• Therefore, overnight cultures of pJUMP28-1A, pJUMP29-1A, BBa_J23110, BBa_J23100, BBa_J23101, and BBa_J23104 will be made
• 50 mL liquid cultures were made, and then split equally between two Falcon tubes

• Made spread plates of pJUMP28-1A (with 1 µL liquid culture) in E. coli, using some unlabelled -80 C frozen competent cells
• These cells had a very low transformation efficiency, and recovery with SOC is more effective
• Therefore, this will be reattempted with pUC19 to determine if pJUMP is causing issues, or if the issue lies with the competent cells
• Made up new competent cells, and subsequently stored on glycerol at -80 C, following this protocol

• Carried out PCR on all growth slowers, as well as pET28-a (to add BsaI flanking sites to it)
• Digested the following with the restriction enzymes:

J23100 + GFP	BsaI, NcoI
J231100 + GFP	BsaI, NcoI
J23101	BsaI
J23104	BsaI
pJUMP28-1A
pJUMP29-1A

• 1 µL of enzyme was used in each digest

• Nanodrop results shown in the table below: ('!!' denotes an unexpected result, with two '!!' indicating errors at both 260/230 and 260/280)

Nanodrop results
gp240	31.066
gp0.7	61.699 (260/230!)
gp79	48.204 !!
Alc	51.235 !!
gp2	22.393 !!
gp104	30.070 !!
AsiA	48.589 (260/230!)
pET	47.731 !!
pJUMP28-1A (top)	6.623 !!
pJUMP28-1A (top 2)	3.829 !!
pET (top)	6.034 !!
pET (bottom)	8.085 !!

• Made a 1% agarose gel with the previous day's digests and PCR products
• Most gels were expected
• The gel extractions had almost no DNA present, however
• The PCR was left over the weekend, holding at 4 C for all growth slowers and pET28-a

• Digester the three BBa_J23100 constructs

Component	110-7	110-8	110-9	pJUMP28	pJUMP29
DNA	6.8	11.5	4.6	24.3	6.3
NcoI	1	1	1	1	1
BsaI	1	1	1	1	1
rCutsmart	5	5	5	5	5
Water	36.2	31.5	38.4	18.7	36.3

All volumes in table above in µL

• Ran growth slowers and pET28-a on a 1% agarose gel, with PCR being completed on gel extractions over the weekend
• pJUMP29-1A was not present in the gels

• Redid PCR of growth slowers that had failed (gp0.7, gp2), as well as pET28-a
• Ran 1 µL of each PCR product on a 1% agarose gel (1 µL DNA, 1.5 µL sterile water, 0.5 µL loading dye)
• The remaining 49 µL was used in PCR cleanup, leading to a final volume of 30 µL
• Transformed pUC into competent E. coli cells (1 µL of 214 ng/µL), to allow comparison
• Set up overnight cultures of pJUMP29-1A, to allow PCR after the mini-prep is complete
• Set up PCR of vsfGFP (by resuspending primers and gBlocks) as well as sfGFP (resuspended primers and miniprepped pJUMP28-1A (which was used as a template))

Gene	FWD Primer Volume	RVS Primer Volume	Master Mix Volume	Template DNA Mass	Template DNA Volume	Wo’ah Volume
vsfGFP	0.25 µL	0.25 µL	25 µL	5ng	0.5 µL	24 µL
sfGFP	0.25 µL	0.25 µL	25 µL	8ng	0.2 µL	24.3 µL

• The PCR of pET and the growth slowers was successful

• From the gel above, the success of the PCR of vsfGFP can be seen, but the PCR of sfGFP did not work

• Carried out restriction digests of pET and growth slowers

Component	pET	0.7	2	240	104	Alc	AsiA	79
DNA	12.7	7.9	13.9	32.2	33.2	19.5	20.5	20.7
BsaI	1	1	1	1	1	1	1	1
rCutsmart	5	5	5	5	5	5	5	5
Water	31.3	36.1	30.1	11.8	10.8	24.5	23.5	23.3

• From 11-8-23, we used gp240, gp104, gp79, Alc, AsiA, gp0.7, gp2, and pET28
• Carried out the following ligations:

Growth Slower	Volume pET, µL	Concentration growth slower	Volume growth slower in digest, µL	Volume of ligation, µL	Volume water, µL
Gp2	5.68	71.62	13.90	8.14	3.18
AsiA	5.68	48.59	20.50	5.52	5.80
Gp240	5.68	31.07	32.20	3.53	7.79
Gp0.7	5.68	125.68	7.90	14.28	-2.96
Gp79	5.68	48.20	20.70	5.48	5.84
Gp104	5.68	30.07	33.20	3.42	7.90
Alc	5.68	51.24	19.50	5.82	5.50

• No water was added to gp0.7, due to the calculated volume of water being negative

• Carried out transformations:

Assigned Number	DNA	DNA vol (µL)	Competent Cell Batch	Volume competent cells, µL
1	GP2	8	Max	50
2	Asia	8	Max	50
3	GP240	8	Max	50
4	GP0.7	8	Max	50
5	GP104	8	Max	50
6	GP79	8	Max	50
7	Alc	8	Max	50
8a	pUC19 (1ng /µL)	1	Max	50
8b	pUC19 (1ng /µL)	1	Katie	50
9	pJUMP28 (T1)	1	Katie	50

• Used different masses of DNA, as the concentration of these samples had not been determined with the Nanodrop, due to protein contamination
• None of these transformations were successful. Therefore, these will be reattempted with new competent cells
• Ligation:
• E. coli grown on eight ampicillin and two kanamycin plates, all at 50 mg /mL
• vsfGFP concentration determined with Nanodrop, and recorded results in gene progress spreadsheet
• Ensured there was adequate pET28-1A. There is not, so more will be synthesised by PCR
• PCR of pET28-1A:

Backbone	FWD Primer Volume, µL	RVS Primer Volume, µL	Master Mix Volume, µL	Template DNA Mass (ng)	Template DNA Volume, µL	Water Volume, µL
pET28-1A	0.25	0.25	25	~6.2	1	23.5

• The transformations from 16-8-23 were redone, with new competent cells
• Halved everything, as per the protocol

Assigned Number	DNA	DNA vol, µL	Volume of competent cells, µL
1	GP2	4	25
2	Asia	4	25
3	GP240	4	25
4	GP0.7	4	25
5	GP104	4	25
6	GP79	4	25
7	Alc	4	25
8 +ve control	pUC19 (1ng /µL)	0.5	25

• None of these transformations were successful, due to pET28-1A having ampicillin resistance, and the BsaI restriction site being in the ampR gene
• pUC transformation was successful! The cells went red, so they are definitely competent
• Therefore, the cells were prevented from growing, due to the confusion with antibiotic resistances
• PCR cleanup of pET28-1A completed, then digested with BsaI
• vsfGFP digested with BsaI
• Carried out restriction digests:

Component	vsfGFP	sfGFP	pET	pET
DNA	4.8	21.3	7.7	9.5
BsaI	1	1	1	1
rCutsmart	5	5	5	5
Water	39.2	22.7	36.3	34.5

• The restriction digests were incubated at 37 C for 1 hour
• Ligated GFP with pET28-1A

Insert	Vol (ul)	pET vol (ul)	Ligation Buffer (ul)	Ligase (ul)	Water (ul)
sfGFP	1.60	0.78	4	2	31.6
vsGFP	0.36	0.78	4	2	32.9

• Nanodrop results from before the ligation:

vsfGFP	207.717 (260/230!)
sf GFP	47.008 (260/230!)- didn't work on the gel either

• PCRs of pET28-1A were successful; a gel electrophoresis was run to confirm:

• Received suggestions from our PI, Dr. Andy Hitchcock, re. the PCR of pET28-1A:
• Extension time of 6 minutes at 60 C, 1 µL of 10 mM primers, 0.5 µL pET28-1A, 25 µL q5 Master Mix, and 22.5 µL sterile water
• Completed the PCR of pET28-1A:

Backbone	FWD Primer Volume, µL	RVS Primer Volume, µL	Master Mix Volume, µL	Template DNA Mass, ng	Template DNA, µL	Water Volume, µL
pET28-1A (Andy’s)	0.25	0.25	25	~unknown	0.5	24
pET28-1A (Andy’s)	0.25	0.25	25	~unknown	0.5	24

• Set up overnight cultures to produce competent Lemo BL21 and DH5α E. coli
• Completed colony PCR of vsfGFP, with the following volumes per reaction:

PCRBIO Taq Mix Red
Reagent	Initial Conc.	Vol. per reaction, µL	Final conc.
PCRBIO Taq Mix Red MasterMix	2×	5	1×
Fwd Primer	10 µM	0.2	0.2 µM
Rev Primer	10 µM	0.2	0.2 µM
H2O, nuclease free	-	2.6	-
Template	-	2	-

• Used 2× PCRBIO Taq Mix Red
• Took colonies with a pipette tip, scratching them on to a record-keeping plate, then dipping the tip in to the mix with the other reagents
• Used 4.6 µL of sterile water (2.6 µL + 2.0 µL of the template)

• Ran colony PCR with the ligations of pET28-1A, vsfGFP, and growth slowers
• Made up a mix of Master Mix, primers, and sterile water to 800 µL, for 32 PCRs

Component	Volume, µL
Master Mix	400
Fwd Primer	50
Rev Primer	50
Sterile Water	300

• Set up overnight cultures of BL21 and DH5α E. coli, to allow more competent cells to be made tomorrow

• Took optical densities at 600 nm, of the overnight cultures:

OD600 of overnight cultures
BL21	0.57
DH5α	0.52

• Set up three colony PCR gels
• Made new competent cells, using the Perak protocol
• Redid PCRs using the same volumes as 21-8-23, for more screening of the growth slowers
• Produced scratch plates, with kanamycin and agar

<\

• Digested gp240 and gp104 with BsaI, at 37 C for 1 hours

DNA	DNA vol, µL	Vol of 10X cut smart buffer, µL	BsaI vol., µL	Water vol., µL
gp240	20	5	1	24
gp104	20	5	1	24

• Carried out transformations of new competent cells, testing the transformation efficiency of the competent DH5α and BL21 E. coli
• Used pUC19 and pJUMP28, 1 µL of each
• Made two kanamycin (50 µL Kan into 50 mL LB agar) and two ampicillin (50 µL ampicillin into 50 mL agar) plates

• Ordered primers, gBlocks, reagents, pipette tips
• Recorded the concentration of DNA from the digested products of gp104 and gp240, with the Nanodrop
• Ligated pET28-1A and gp104, gp240

Growth Slower (GS)	Vol. GS DNA, µL	Vol. pET28 DNA, µL	Vol. Buffer, µL	Vol. Ligase, µL	Vol. Nuclease-Free Water, µL
GP240	0.348	2	2	1	14.652
GP104	0.4266	2	2	1	14.5734

• Miniprepped the growth slowing constructs
• Transformed the growth slowing constructs in to BL21 E. coli, following the iGEM protocol
• vsfGFP constructs were sent away for Sanger sequencing
• Ran agarose gel with the colony PCR products from 24-8-23
• Ordered Anderson promoter gBlocks, Rubidium chloride, and primers

Lane	Name	Visible	Length (bp)	Position (bp)	Correct?
1	100 bp Ladder	Yes	–	–	–
2	GP2 – 6	Yes	195 + 165 = 360	>2000	No
3	GP2 – 7	No	360	–	–
4	GP2 – 8	No	360	–	–
5	GP2 – 9	No	360	–	–
6	GP2 – 10	No	360	–	–
7	GP0.7 – 6	No	354 + 165 = 519	–	–
8	GP0.7 – 7	No	519	–	–
9	GP0.7 – 8	No	519	–	–
10	GP0.7 – 9	No	519	–	–
11	GP0.7 – 10	No	519	–	–
12	–	–	–	–	–
13	–	–	–	–	–
14	GP79 – 6	Yes	249 + 165 = 414	>2000	No
15	GP79 – 7	Yes	414	350 < X < 400	Possible
16	GP79 – 8	No	414	–	–
17	GP79 – 9	Yes	414	350 < X < 400	Possible
18	GP79 – 10	No	414	–	–
19	100 bp Ladder	–	–	–	–
20	1kb Plus Ladder	–	–	–	–

Lane	Name	Visible	Length (bp)	Position (bp)	Correct?
1	100 bp Ladder	Yes	–	–	–
2	ASIA – 6	Yes	273 + 165 = 438	>2000	No
3	ASIA – 7	No	438	>2000	–
4	ASIA – 8	No	438	>2000	–
5	ASIA – 9	No	438	–	–
6	ASIA – 10	No	438	–	–
7	Alc – 7	No	504 + 165 = 669	>2000	No
8	Alc – 8	Yes	669	300 < X < 400	No
9	Alc – 9	No	669	–	–
10	Alc – 10	No	669	850 < X < 1000	No
11	Alc – 12	No	669	–	–
12	–	–	–	–	–
13	1kb Plus Ladder	–	–	–	–

Created overnight cultures in DH5α with kan 50 ng /µL, 10 mL LB of:

• 7: Colony 2a from a spread plate.
• Gp2: Colony 2a from a spread plate.
• Gp79: Cluster 1 from scratch plate created on 22/8
• AsiA: Cluster 3 from scratch plate created on 22/8
• Alc: Cluster 3 from scratch plate created on 22/8

The plate with supposedly Alc was fluorescent so a mixup was suspected.

Transferred all the glycerol stock of vsfGFP transformants into 10 mL LB and 5 μL of 50 mg /mL chloramphenicol.

Repeated ligations of gp240 and gp104 into pET28 due to suspected fault in procedures on 24/08. Corrected protocol below:

• Reaction mixture was incubated at room temperature for 30 minutes
• Reaction mixture was incubated at 65 C for 10 minutes
• Reaction mixture was incubated at 16 C or 4 C for ligation

• Miniprepped the overnight cultures from 29/08.
• Transformed extracted DNA from above into BL21 LEMO
• Recreated a glycerol stock of vsGFP
• Remade competent LEMO BL21 cells using the Perak protocol.
• Diluted the overnight culture of BL21 with 400 mL LB to adjust the OD
• Prepared TFB1 and TFB2 mixture using Perak protocol and stored in 4 C

• Miniprepped yesterday’s overnight cultures and transformed them into BL21 LEMO. Could use the DNA and cells we already have (?) 
• Performed colony PCR with gp240 and gp104 DH5α transformants from spread plates
• Ran gel electrophoresis of PCR products to select colonies to make overnight LB broth cultures (50 mg /mL Kan) of.
• Made glycerol stock of pET-vsfGFP-2 transformed DH5α.
• Made new competent BL21 Lemo cells using the Perak protocol.
• Received Sanger sequencing data for vsfGFP-pET28

Reaction mixture for 20 colony PCRs:

• 100μL 2X taq master mix
• 4μL of each primer (pET fwd and pET rev, 10μM)
• 92μL nuclease free H2O

Gel Results:

To verify the growth slowers constructs as correct, below are sent for sequencing:

• Gp79 constructed on 22/8, (BL21) plated on 31/8.
• Alc constructed on 22/8, (BL21) plated on 31/8
• AsiA constructed on 22/8 (likely failed, band at ~850bp marker with expected size at ~450bp) plated on 31/8
• GP2 constructed on 22/8,(BL21) plated on 31/8.
• 7 constructed on 22/8, (possibly failed, band at ~300bp marker with expected size at [slightly larger]), plated on 31/8 (BL21).
• Discarded the wrong ones mini prepped for 240.

Gel results:

Checkmarks denote that the band is at expected position. Numbers index selected colony/cluster from spread/scratch plate.

• Prepared overnight LB broth cultures (50mg/mL Kan) of growth slowers with pET28 backbone in DH5⍺ for glycerol stock and for sequencing.
• Miniprepped successfully transformed colonies (GP104 16°C- 4 and 6, GP240 4°C- 2, 16°C-1) and transformed the extracted gp104 and gp240 into BL21.
• Prepared glycerol stocks of transformed BL21.
• To send all constructs for Sanger sequencing, excluding two growth slowers (gp104 and gp240) that have not been miniprepped yet.
• Prepared overnight LB broth cultures (50mg/mL Kan) of the above two growth slowers
• Performed colony PCRs of the dodgy growth slowers.
• Performed colony PCR to screen 12 more colonies of the constructs with AsiA and gp0.7.

• Extension time formula used: 1 minute per kb
• The size of the gp0.7 construct is 354 +165bp (from backbone) = 519bp. (31 sec extension time)
• The size of the Alc construct is 504 +165bp (from backbone) = 669b. (40 sec extension time)

Gel results:

• Band sizes are smaller than expected, the constructs will be sent for Sanger sequencing for verification.
• Diluted newly arrived GBlock orders into 1 ng /μL stocks.

• Miniprepped the following samples: AsiA (colony 19), GP0.7 (colonies 16 and 17), gp104 (16°C) (colony 6), gp104 (16°C) (colony 4), gp240 (4°C) (colony 2), gp240 (16°C) (colony 1).
• PCR minipreps above.
• Run the PCR products on a gel. Predictions and actual sizes below:

DNA	Predicted size (bp)	Observed Size (bp)
J23100 + vsfGFP	1266	~1250
J23106 + vsfGFP	1266	~1350
J23110 + vsfGFP	1266	~1250
J23114 + vsfGFP	1266	~1250
J23119 + vsfGFP	1266	~1350
Bmo Part 1	2099	/
Bmo part 2	569	/
Bmo part 3	1207	~1500
pET28 Anderson TU BB	5156	~5500
pET28 Bmo BB	5205	~4500

• Performed PCR clean up for viable products according to gel results
• Digested cleaned up DNA with BsaI for Golden Gate Assembly
• Performed Golden Gate assembly

Made glycerol stock of following growth slowers in DH5⍺:

• 7
• Gp2
• Gp79
• AsiA

The plate with supposed Alc construct transformants was determined to be contaminated by vsfGFP due to fluorescent colonies.

Discarded minipreps from Brooks’ pET and the contaminated Alc plate.

Repeated gel electrophoresis for PCR of Bmo part 1 and Bmo part 2

Gel results:

• PCR amplified Alc to prepare for insertion into pET28.
• Sent growth slower-transformed colonies’ (gp0.7, gp2, AsiA, Alc, gp79) DNA for Sanger sequencing.
  • Alc is suspected to be vsfGFP, gp0.7 and AsiA are suspected to be unknown incorrect sequences

• Reconstructed pET28-Alc plasmid with PCR product from 05/09 and transformed it into DH5⍺.Transformants are plated on 50mg/mL Kan LB agar.
• Transformed gp104 and gp240 and the extra colonies from gp0.7 and AsiA and plated them on 50mg/mL Kan LB agar.
• Digested the 5 amplified Anderson promoter GBlocks with BsaI and inserted into pET28, constructs transformed into (strain) and plated on 50 mg/mL Kan LB agar.
• Repeated PCR for Bmo parts 1 and 2.
• Ran gel electrophoresis to check Bmo1, Bmo2, and Alc PCR products. Only the last showed expected results:

Ran gel electrophoresis to check Bmo1 and Bmo2 stocks, results below were as expected:

• Colonies chosen for colony PCRs of Anderson promoter-GFP transformants:

Anderson TU in pET28	1	2	3
J23100	Glow, small	Weak glow, small	Not glow, big
J23110	Glow, small	Not glow, medium	Not, glow, big
J23106	Glow, small	Not glow, big	Glow, small
J23119	Weakly glow, small	Not glow, medium	Glow, big
J23114	Glow, big	Glow, big	Not glow, big

From gel, PCR products formed without the addition of template and all lanes lack distinct bands. This suggests that the primers are binding non-specifically, possibly amongst themselves. Redesigning primers will be necessary to remove the formation of secondary structures.

Primers used: J23100+vsfGFP+BsaI_Fwd and pET Rev= size of 1312bp

• Created plates with 50mg/mL(?) Kan
• Planned the 96 well plate reader experiments

Noticed that the wrong primers had been used for the growth slowers’ PCR, where the wrong overhang was added.

Ran PCR of the growth slowers with the correct primers to add the proper BsaI overhang.

PCR of growth slowers:

Prepared cultures to be at OD 0.05 for the plate reading test (vsfGFP) with Dr Lidbury.

PCR purifications of growth slowers and pET28

Restriction mixture for digest of purified PCR products

Component	104	gp2	gp79	Alc	AsiA	gp0.7
DNA (μL)	10.6	10.2	7.9	6.4	11.9	6.6
BsaI (μL)	1	1	1	1	1	1
rCutsmart (μL)	5	5	5	5	5	5
H2O (μL)	33.4	33.8	35.1	37.6	32.1	37.4

Component	240	pET (1)	pET (2)	pET (3)	pET (4)
DNA (μL)	7.1	4.9	8.2	5.8	4.6
BsaI (μL)	1	1	1	1	1
rCutsmart (μL)	5	5	5	5	5
H2O (μL)	36.9	39.1	35.8	38.2	39.4

Made overnight LB broth culture (50mg/mL Kan + ? Cmf) with J23100-vsfGFP

Digests with DpnI:

Component	Pet28 AND	pET28 BMO	GSpET 2	GSpET1
DNA (μL)	25.6	44	8.2	21.7
DpnI (μL)	1	1 (also  BsaI)	1	1
BsaI (μL)	1	1	1	1
rCutsmart (μL)	5	5	5	5
H2O (μL)	17.4	0	34.8	21.3

Ran PCR for pET-vsfGFP with primers pET28-BsaI fwd and pET28-BsaI-lacI rev to add BMO overhangs.

Template DNA: pET-vsfGFP (2.1 µg /µL added 1 µL)

Note: With the pET28-BsaI-LacI rev primer resuspended but without a diluted stock, pET28 for BMO used above may be faulty. All primer dilutions are done to 10 mM unless otherwise stated. All primer dissolving is done to 100 µM.

• Run on gels: colony PCRs and digests of growth slowers and pETs from in final experiments.
• Insert growth slowers to pET28 backbone
• For plate readings: measure absorbance with spectrophotometer near the laminar flow hood on bay 5 for consistency.
• Emailed Andy to meet him 14/09 as he is unavailable on Friday
• Fill in/submit safety forms
• Discarded old digests and repeated them properly as reaction mixtures lacked one enzyme
• Due to insufficient purified DNA, the reaction was scaled down to a total volume of 27.5 μL

Components	Volume (μL)
Pet28 AND	14
DpnI	0.55
BsaI	0.55
rCutsmart	2.75
H2O	9.65

• Transformed ligations into DH5α and plated on 50 mg /mL Kan LB agar
• Gave plate reading data to Berardo and Fyona
• Created a 96 well plate with IPTG ranges between 0-4 µM

Checked purity of previously miniprepped DNA from ligation colonies.

Note: DNA was loaded before completely filling the tank with TAE buffer, hence the misshapen bands.

Construct in colony	T7 mastermix (μL)	10uM forward primer pET fwd (μL)	10uM reverse primer pET rev (μL)	Nuclease free H2O (Make volume up to 10μL)	Amount of each primer added (μL)
Gp79 in pET28	5	0.2	0.2	4.6	0.2
Gp240- in pET28	5	0.2	0.2	4.6	0.2
Alc-pET28	5	0.2	0.2	4.6	0.2
Gp79- pET28	5	0.2	0.2	4.6	0.2
Gp104- pET28	5	0.2	0.2	4.6	0.2
Gp0.7- pET28	5	0.2	0.2	4.6	0.2
Gp2- pET28	5	0.2	0.2	4.6	0.2
AsiA- pET28	5	0.2	0.2	4.6	0.2

Ran gel electrophoresis on colony PCRs. No bands were formed.

• Did a positive control test of the primers thought to be degraded as the reason for the lack of bands. The test confirmed them to be intact. It was concluded that the ligation procedure was the source of error.
• Repeated the transformations with existing growth slower constructs in pET28 and repeated the colony PCRs (taq mastermix) .
• Colony PCRs using Taq master mix

Reaction mixture:

Reagent	Vol (μL)
Taq master mix 2x	40
Primer Fwd	1.6
Primer Rev	1.6
H2O	17.2

Each colony was added to 10 µL of the mixture for PCR

Prepared overnight LB broth cultures (50 mg /mL Kan) of the colonies selected for PCR

To the scratch plates made on 19/08, added the following colonies

• Gp240: 4,5,6
• Gp104: 4,5,6
• Gp0.7: 4,5,6
• Alc: 4,5,6
• Gp2: 4,5,6
• AsiA: 1,2,3
• Gp79: 4,5,6

Other colonies (streaks) on these plates have been deemed unsuccessful by colony PCR on 19/08.

• Prepared overnight LB broth culture (various antibiotics) of vsfGFP, pET28 and pJUMP28 in DH5α
• Performed colony PCRs and made overnight LB broth cultures (various antibiotics) of the new ligation transformations in DH5α.
• Verified ligation success with gel electrophoresis
• PCR amplified the Anderson promoter DNA, then checked with gel electrophoresis.
• Ligated more growth slower genes to backbone plasmids

The primers that were used for PCR mixtures for wells 2-8 are Primer for Bmo colony PCR_Fwd and pET_Rvs.

• Ran colony PCR products from the previous day on a gel, results were disappointing.
• Collected pET28 from Dr Hitchcock
• Transformed DH5α with the new pET28
• Plated transformations on 50 mg /mL Kan LB agar and made overnight LB broth culture with 50 mg /mL Kan
• PCR amplify new GBlocks of the anderson-vsfGFPs:

GBlock	Primer (10uM)Fwd	Primer (10uM)Rvs	Primer fwd (μL)	Primer Rvs (μL)	Template (μL)	Q5 master mix (μL)	Nuclease Free H2O (μL)
J23100-vsfGFP	J23100+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	1	25	19
J23106+ vsfGFP	J23106+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	1	25	19
J23110+ vsfGFP	J23110+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	1	25	19
J23114+vsfGFP	J23110+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	1	25	19
J23119+ vsfGFP	J23119+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	1	25	1
Control	J23100+vsfGFP +BsaI fwd	Anderson + vsfGFP+ BsaI rev	2.5	2.5	0	25	20

• Backbone (pET28 GS) cleanup was nanodropped to show subpar concentration and purity
• Ran gel electrophoresis to verify amplified GBlock size. First gel with Anderson promoter GBlocks and second gel with growth slower GBlocks.

Gel results:

• GP2 and GP0.7 did not form distinct bands.
• Only J23119 + vsfGFP among the Anderson GBlocks formed a distinct band.

Transformed pET28 into DH5α to amplify the two versions of the backbone we need out of it.
Checked the amplified growth slower GBlocks and the Anderson + vsfGFP GBlocks.

Size verification for GBlocks and backbone plasmids.

Ran gel electrophoresis to check Anderson+vsfGFP extracted from colonies.

Primers used: forward primer that binds specifically to each individual Anderson Promoter and the reverse colony PCR primer that binds to pET28a.

• With Brooks’ PCR clean up/Gel extraction kit used 20 μL elution buffer and left for 5 mins.
• Cleaned up alc, asia, gp79, gp0.7, gp2, gp104, gp240, pET28 GS (x2)
• Ran on 0.75% small agarose gel for 20 mins at 110V the pET28 GS cleaned up (20μL of pET28 in each tube+3.5μL of purple ladder NEB).
• Nanodrop results for the GSs: good purity, all beautiful graphs with peaks at 260nm :)

Growth slower	Concentration (ng /μL)
gp0.7	114.143
gp2	117.372
gp79	88.196
gp104	157.023
gp240	138.398
Alc	161.506
AsiA	152.961

A BsaI-digest was then performed to prepare overhangs for ligation

• PCR and electrophoresis were done to test for desired DNA from J23119+vsfGFP+pET28 colony minipreps.

Lane 1	Lane 2	Lane 3
1kb plus ladder - Invitrogen.	J23119+vsfGFP+pET28-1.	J23119+vsfGFP+pET28-3.

• Completed BsaI digests of the growth slowers.
• Gel extraction of pET28: 1= 0.17g (added 510 μL dissolving buffer), 0.22g (660 μL) and 2=0.27g (810 μL), 0.13g (390 μL)
• Amplified pET28 GS ×4 via PCR with pET rev and pET fwd.
• PCR cleaned up using Brook’s kit.

BsaI and DnpI digest of the following:

pET28 digests	pET28 conc (ng/μL)	pET28 vol (μL)	rCutSmartBuffer (μL)	H2O (μL)	BsaI (μL)	DnpI (μL)
1	102.5	9.8	5	33.2	1	1
2	82.5	12.1	5	30.9	1	1
3	61.9	16.1	5	26.9	1	1
4	92.2	10.8	5	32.2	1	1
efasGFP	34.2	29.2	5	14.8	1	1

• pJUMP28 miniprep concentration= 42.616 ng /μL
• efasGFP miniprep concentration = 34.224 ng /μL
• The first set of GS backbones from PCR produced smeared lanes on gel electrophoresis
• Performed another four GS backbone PCRs and clean up

Diagnostic gel with 4 GS backbones:

1% gel for extracts, midori green, and purple 6× loading dye (to each 50 µL sample added 9 µL dye)

1	2	3	4	5	6	7	8	9	10
1kb plus	pet1	pet1	pet2	pet2	pet3	pet3	pet4	pet4	1kb plus

• Gel extracted pET28a-1, 2, 3 and 4 (lanes 2-9), nanodrop results were subpar (18 ng /μL with very bad ratios and graph curves were off by a wide margin, low concentration and high contamination).
• Cleaned up remaining digested pET28 from 25/09 to prepare for ligations tomorrow.
• Remade overnight LB broth culture (50 mg /mL Kan) of DH5α with pET28 generated using pETfwd and pET28 rev.

• Cleaned up PCR products of sfGFP using our kit.
• Digested sfGFP, amounts on the 25th digest spreadsheet.
• Ran four repeats of pET28 GS backbone PCRs.

• Cleaned (eluting in 20 μL), then digested with BsaI and DpnI and cleaned again (eluting in 25 μL) the pET28 GS PCR products.
  • They had a relatively low yield (in future after PCR, we need to combine them and run them through the same clean up column.)
  • Made a final cleaning step to combine and concentrate all four pET28 GS solutions
• Ran 7 μL on a gel with 7 μL loading dye and the band was bright, it was decided to go ahead with ligations into the pET28 GS.
• Ligation mixture: 1 μL insert (gp79, gp104, gp240, gp0.7, Alc, AsiA), 1 μL template, 1.1 μL buffer, 1 μL ligase, 5.9 μL H2O
• Transformed ligations into DH5α and then plated on 50 mg /mL Kan LB agar and incubated at 37°C overnight.

• Cleaned (eluted in 50 μL- should use 25 μL in future) sfGFP from PCR and ran 10 μL on 1% agarose gel with 10 μL loading dye.
• Created the following 10 μL overnight LB broth cultures that are in E. coli BL21:
  • 1 Ampicillin (10 μL) for efasGFP
  • 5 kanamycin (10 μL) and chloramphenicol (5 μL) for Anderson-vsfGFPs and pJUMP28
  • 1 kanamycin for pET28 (This may be in DH5α)
• Ligated sfGFP into pET28: 1 μL sfGFP, 1 μL pET28, 1.1 μL buffer, 1 μL ligase, 5.9 μL MQ H2O

• All the colonies of sfGFP in pET28 fluoresced, so colony 3 was miniprepped

• The candidate colonies to miniprep for transformation into BL21 from the colony PCR are:
  • Gp79-1 (right size in PCR gel later on today)
  • AsiA-1 (right size in PCR gel later on today)
  • Gp2 - 2 (not right size in PCR gel later on today)
• Miniprepped efasGFP.
  • Concentration: 38.040ng/uL
• Digested efasGFP (with only BsaI, no DpnI)
• Ran the result on a gel
  • Could not be extracted from gel as all the DNA stuck to the well.
  • Brooks’ theory is that that is genomic DNA and not plasmid DNA. To test this, 3 μL of the original miniprepped DNA was ran on a gel to yield the same result, so it is likely there was no plasmid DNA in the miniprep.
  • Brooks will run on 01/10/23 some more efasGFP DNA on a gel to check it.
  • Made an overnight 10 mL LB broth culture (50 mg /mL Amp) of DH5α with efasGFP DNA to miniprep more.
• The transformations Katie did on 28/09 were all successful except for pJUMP28. pET28 in BL21 and in DH5α all worked. These plates are in Brooks’ 4 C room.
• PCR of all colony miniprep DNA.
  • Strand 1
    • GP2: 4,5,6
    • 7: 3,4,5
    • GP79: 1,2
  • Strand 2
    • GP79: 3
    • GP104: 1,2,3
    • GP240: 4,5,6
  • Strand 3
    • Alc: 1,2,3
    • AsiA: 1,2,3
  • The first picture has all the colony DNA of strands 1 and 2 plus Alc of strand 3, in order.

• The second picture has the two joined wells where the efasGFP digest was meant to be extracted from. All the DNA remained in the wells. At the end there’s DNA of the three AsiA colonies that look about the right size.

• The PCR DNA product that looks right is from:
  • gp2 colony 4
  • None of gp0.7
  • gp79 colonies 1,2,3
  • gp104 colonies 1,2,3
  • gp240 colony 5
  • Alc colony 1
  • AsiA colonies 1,2,3

• Prepared 5x LB broth culture media, with 5 mL LB, 5 μL Kanamycin stock (50 mg /mL)
• Kan defrosted at room temp, then refrozen in iGEM freezer box (primers and G blocks)
• Prepared 8× agar plates in aseptic condition by Bunsen flame, 25 μL kan, 50 μL chloramphenicol, using agar from 28/9, reliquified by microwave
• Uploaded Anderson sequencing data to Benchling
• 1M IPTG solution transferred from Perak freezer to iGEM freezer box in Andy’s lab for plate reader preparation tomorrow
  • Tubes for this labelled and stored in ice box on top shelf of bench
• Transformed sfGFP and all growth slowers except for gp0.7 into LEMO (BL21).
• Ran five more gp0.7 colony PCRs and incubated their respective overnight LB broth cultures (50mg/mL Kan).
• Made efasGFP overnight LB broth culture (50mg/mL Amp) and another overnight (antibiotic) required for the Anderson plate reading experiment.

• Prepared plate reading for the Anderson promoters
• Made overnight LB broth cultures (50mg/mL Kan) of all the prepped growth slowers

• Prepared growth slowers for Sanger sequencing
• Checked for efasGFP colonies formed on plate from 3/10?
• Miniprep the overnight culture from 3/10

• Last sequencing results received (used GATC Services from Eurofins Genomics).
• The sequencing of the GS and sfGFP constructs used primers that bound to the T7 pLac promoter.
• The sequencing of the Anderson constructs used a primer that bound 167 bases upstream from the Anderson promoter.
• PDFs with the sequence alignments have been saved here. Results are as follows:

GS	Successful?
Alc	No
AsiA	Yes
gp0.7	No
gp2	Yes
gp79	Yes
gp104	Yes
gp240	Yes
sfGFP	Yes
J23100-vsfGFP	No
J23106-vsfGFP	Yes
J23110-vsfGFP	Yes
J23114-vsfGFP	Yes