Experiments

Week 1

We prepared competent DH5α cells according to the protocol Chemical competent cells. These were to be used in upcoming experiments.

We also received primers ordered and prepared these. gBlocks were ordered from IDT, and these were PCR amplified using Taq polymerase following the protocol from Ampliqon. Only weak bands were seen on the gel, so it was decided to do the experiment again.

Week 2

gBlocks were successfully PCR amplified using Taq polymerase following the protocol from Ampliqon. Products were stored in the freezer.

We prepared two of our backbones by linearizing pUC19. This was done following the protocol for X7 PCR using Phusion buffer instead of CxL. Fragments were checked on a gel and purified from overnight cultures the following day. The plasmid purification was conducted following the protocol from Invitrogen.

To obtain the second plasmid we needed for our experiments, we transformed pACYC184 into DH5α. Growth was obtained in LB supplemented with chloramphenicol. Concentrations of pACYC184 were too low to be visualized on a gel with EtBr. It was concluded that a PCR had to be done. After 2 days of growth, we were able to purify the plasmids using the Monarch® Plasmid Miniprep Kit from NEB. The obtained concentrations were rather low, so PCR amplification was conducted.

DH5α was transformed with GFP tet-on and mCherry reporter. Transformants were plated on LB supplemented with kanamycin. Controls were made in duplicates as follows: DH5α plated directly on LB with kanamycin, and DH5α plated directly on LB. Growth occurred for DH5α on LB and transformed DH5α on LB with kanamycin. The transformation was considered successful.

We also wanted to test the GFP tet-on system in DH5α. We inoculated 1 colony in 1 mL LB supplemented with tetracycline. It was left to incubate overnight at 37°C.

PCR was conducted on our 6 TMS’s (Theo1, Theo2, Kan, Neo, GFP, Mn) following the protocol for X7 PCR.

Week 3

Troubleshooting PCRs by testing three different X7 polymerases on the TMS aptamer with GFP using appropriate primers. The protocol for X7 PCR was followed. Buffer used was NEB 2x mastermix. Only faint bands were obtained.

First attempt to make competent BL21(DE3) cells was conducted following Chemical competent cells. Due to low growth, this was postponed to next week.

Initial fluorescence measurement of ordered plasmids containing mCherry and GFP-teton. Inducers for the two plasmids were arabinose and tetracycline, respectfully. Different concentrations of the inducers were added:

The strains harboring the plasmids were tested on Fluorescence Spectrometer JASCO model number FP-8500 with GFP emission measured by excitation at 395 nm, mCherry emission measured by excitation at 561 nm. The presence of GFP was shown, but the presence of mCherry could not be seen. This, we found out was because we were using D-arabinose, which is unable to induce the araBAD promoter.

First successful USER cloning of the TMS-GFP into pUC19 (pU07) following the protocol USER cloning. These were tested the following week.

Week 4

This week we prioritized: Troubleshooting PCRs, USER cloning of our plasmid, and looking into further fluorescence measurements.

New competent BL21(DE3) were made and their competency checked using pUC19 control from NEB. Growth of transformed strains was not obtained on LB supplemented with ampicillin plates, but this was also not achieved for our competent DH5α. Growth on LB plates was obtained.

We continued our experiments for mCherry induction by arabinose. Here, we came to understand that our araBAD promoter is only induced by L-arabinose, and we had been using D-arabinose. We fixed the problem and obtained some initial results:

For troubleshooting PCRs, we tried to add DMSO, reduce the primer concentration, and change the annealing temperature. No additional knowledge was achieved from this.

We successfully USER cloned pU08-10, but not pU02. These were checked with colony PCR in the following week.

Week 5

BL21(DE3) competency test by transforming the competent cells with pACYC184, pUC19 (kindly provided by Mogens Kilstrup), and pUC19 (NEB), respectively. A control with MQ was also made and plated onto LB. Transformations were done according to Transformation of E. coli (normal heating times used, and for pACYC184, cells were incubated in 475 μL LB at 37°C for 1 hour). Growth was obtained on all plates, thus, cells were concluded to be competent.

New batch of competent DH5α was made by following Chemical competent cells. Cells were stored in the -80°C freezer.

Our USER constructs, pU08-10, were checked with colony PCR (elongation time of 2.5 min). Overnight cultures were made of the validated strains, and the plasmids were purified using Monarch® Plasmid Miniprep Kit from NEB, and cryostocks were made.

We continued with our PCRs to amplify the following fragments: B01, B03, B07, B11, B12, B13. We used the PCR program with annealing temperature of 57°C and elongation time of 30 s for B01 and B03, and annealing temperature of 56°C and elongation time of 2.5 min for B07, B12, and B13.

PCR troubleshooting: we figured that we had been using an unfit buffer (10x buffer), we then decided to use rCutSmart moving on. Also concluded that 2X Phusion U Hot Start Master Mix from Thermo Fisher could be used.

USER clonings of pU01, pU02, and pU03. The USER cloning was conducted according to USER cloning, and all were grown in LB for 1 hour before plated onto LB supplemented with chloramphenicol and ampicillin. The positive control was pACYC184, and the negative control was the backbone (G03) and MQ. Nice positive and negative controls were obtained, but no colonies on the cloned plates.

Week 6

PCRs of B10, B11, B12, and G03 were redone using rCutSmart as buffer. Also, B03, G05, G06, G08, B07, and B09. Gel extraction was done on B07, G05, G06, G08, and PCR cleanup on B03 and B09. Concentrations and purities were the best for B03 and B09, and moving on we used PCR cleanup instead of gel extraction.

USER clonings of pU01, pU02, and pU03 were redone. We had three versions of the backbone (G03), and USER cloning was conducted with one of each doing equimolar amounts of backbone and fragment. As a positive control pACYC184 was used, and cloning was done with 1 min of heat shock, and incubation in 450 μL LB media for 1 hour before plating onto LB plates supplemented with chloramphenicol.

The cloning did not succeed. We considered if this was due to the plasmid backbone not being opened correctly. A possible reason for this could be plasmid supercoiling for which reason linearization using restriction enzymes would be the best option. Another cause could be the annotated plasmid pACYC184 is not correct.

An experiment was set up to assess the linearized backbone issue. We wanted to check if the previously excised band was just a supercoiled topoisomer. A gel was run with two different PCR mixes for p03 reaction against the non-linearized plasmid. PCRs were done with Phusion MM2X HotStart and X7-CutSmart. Unfortunately, the extension time was not enough for the X7 PCR (2.5min, should have been 3.5min), and on the gel the bands looked more lightweight than they should; nonetheless it was visible that the amplified band ripple, as if there were more PCR products than just the expected one, and the rippling pattern did not correspond to the pattern of the nonlinearized backbones, which is likely only due to just supercoiling. The Phusion reaction did show a band of the right size, which was surrounded up and down by thinner bands corresponding to the nonlinearized backbone supercoiled ripples.

Another Phusion was done, which gave a nice, clear band. This was cut out from the gel and extracted. Due to leftover salts a cleanup was attempted following the second half of a plasmid prep protocol (invitrogen).

We proceeded with USER cloning and did pU01. Colonies were obtained, and transformants were validated using colony PCR. Overnight cultures were prepared for next week.

Week 7

Plasmid pU01, which was made and validated the week before, was purified and a glycerol stock was made. The purified plasmid was additionally checked in a restriction enzyme assay using the restriction enzymes SspI and AdhI. AdhI should generate two fragments of 1054 bp and 4212 bp, and SspI three fragments of 873 bp, 1728 bp, and 2665 bp.

pU01 was transformed into BL21(DE3) to generate a reporter strain, which we used for assessing mCherry production upon arabinose induction. Thus, the strain was induced with varying concentrations of arabinose. The tested concentrations were in g/L: 0, 0.1, 0.2, 0.3, 0.5, 0.7, and 1. Fluorescence increased with increased arabinose concentrations, from 112 to 383, and a fluorescence of 92 for the negative control.

The plasmid backbone pUC19 with kanamycin resistance was constructed with USER cloning using an amplified kanamycin resistance cassette and linearized pUC19 without the ampicillin resistance. The cloned strains were validated with colony PCR and restriction enzyme assay. The restriction enzyme used was SspI, since this generates two distinct fragments (size 374 bp and 2276 bp) and also cuts within the kanamycin resistance cassette. Correct plasmids were purified and glycerol stocks were made.

We attempted double transformations with the USER cloned plasmids pU07-pU10 into BL21(DE3). Due to insufficient transformations a new protocol from Eindhoven iGEM 2014. No successful double transformations were achieved this week.

MIC test of BL21(DE3) with tetracycline. From the results, it seemed cells started to grow at a concentration of 0.2 μg/mL and can survive at concentrations below that.

Week 8

Transformation of USER-cloned plasmids into BL21(DE3). We had trouble getting two plasmids into one strain.

• Unsuccessful transformations tried: BL21(DE3) + pU07 + pU01, BL21(DE3) + pU07 + 2xpU01, BL21(DE3).pU01 + pU07.
• Successful transformations: BL21(DE3).pU01, BL21(DE3), BL21(DE3) + pU07.

To test why the double transformations were unsuccessful, different combinations of transformations were done following the protocol from Eindhoven. Additionally, transformations with a 1 hour induction and a 2 hour induction were conducted. The following transformation combinations were tried: BL21(DE3), BL21(DE3) + pU01 + pU07, BL21(DE3).pU01, BL21(DE3).pU01 + pU07, BL21(DE3).pU07, BL21(DE3).pU07 + pU01. Combinations with “+” are transformed at the same time, and transformations with “.” are done as two rounds of transformation. Transformants were plated on four different plates: LB, LB supplemented with ampicillin, LB supplemented with chloramphenicol, and LB supplemented with ampicillin and chloramphenicol. All transformants were plated on all the different plates, except BL21(DE3) containing both plasmids was not plated onto LB only plates.

One successful transformation of BL21(DE3).pU01.pU07 was obtained. Some varying results were obtained from the 2 hour induction transformants, and these transformations were therefore redone. Alongside, transformations with pU08, pU09, and pU10 were also done.

PCR of B01 and B03. The PCR was done according to X7 PCR with an elongation time of 12 sec, since the two fragments have the size of 175 bp and 173 bp, respectively. The PCR products were cleaned up using Ampliqon PureIT ExoZAP PCR CleanUp and following the instructions given by the manufacturer. The products were verified on a gel and concentrations and purities were evaluated on a Nanodrop.

USER cloning of pU05 and pU06 using G02 as the vector backbone and B01 and B03 in each construct, respectively. The protocol followed was USER cloning. As a negative control MQ was used, and pUC19 (KanR) was used as a positive control. Before plating was done on LB supplemented with kanamycin, the transformants were incubated in LB for 2 hours at 37°C. The following day, all plates contained transformants, and the cloning was therefore attempted again to see if this was just a mistake or something was off with the backbone. The repetition gave the same results, so it was decided to do colony PCR on both rounds of transformants. The cPCR was done using Taq polymerase. These results were not convincing.

We tested tetracycline and arabinose induction of the TMS + mCherry system, the one in BL21(DE3).pU01. Cells were grown until they reached an OD of 0.3, after which they were divided into Eppendorfs and 0.5 g/L arabinose and varying concentrations of tetracycline (listed in table) was added. Cells were incubated at 37°C for 5 hours, and fluorescence was measured on the plate reader BioTek Cytation 5.

After 5 hours, the following results were obtained:

Week 9

Another cPCR of pU05 and pU06 transformants. This time following

X7 PCR

We sent our constructed plasmids to sequencing by Eurofins. The following was sent with their respective primers: pU01, pU07, pU08, pU09, pU10, and pUC19 (kanR). For some of the plasmids, more than one copy was sent.

BL21(DE3).pU01 was made competent, as we considered this a way to increase the chances of successful double transformations. We followed the protocol Chemical competent cells. More competent DH5α was also made following the same protocol.

Another attempt of USER cloning of pU05 and pU06 was done alongside USER clonings of pU02, pU03, and pU04. Cloning was done following USER cloning protocol. Colony PCR was done the following week.

Week 10

Received sequencing data from Eurofins. pU07, pU09 seemed correct.

We did some new colony PCR using Taq polymerase on pU03, pU04, pU05, and pU06. Some of the pU03 had the correct band size, whereas all pU04 had one extra band. It was also concluded that one of the pU06 could be correct, whereas no bands were seen for pU05. Overnight cultures were made of correct plasmids, and these were then purified the following day using the Monarch® Plasmid Miniprep Kit from NEB and following the instructions given by the manufacturer.

More plasmids were sent for sequencing with appropriate primers. These were: pU01, pU03, pU08, and pU10.

This week we also received competent BL21(DE3) (C2527H) from NEB, and with these we initially tried another double transformation. We transformed pU01 and pU08 into the competent cells following the protocol plasmid transformation in E. coli, using 2 μL of each plasmid with a concentration of 2 ng/μL. The cells were heat shocked for 50 s and plated onto LB supplemented with chloramphenicol and ampicillin. The following day plenty of colonies were obtained and these were checked with colony PCR. All checked colonies showed the right band length. Two glycerol stocks were made.

We then proceeded with the other double transformations and did BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10, and BL21(DE3).pU03.pU07. BL21(DE3).pU03.pU07 was not plated since a sudden lack of plates occurred. The others were plated, and transformants were checked the following week.

Week 11

BL21(DE3).pU01.pU09 and BL21(DE3).pU01.pU10 from the week before were checked with colony PCR using Taq polymerase. Overnight cultures for cryostocks were made.

Sequencing data was received. Everything, but pU06, looked as expected.

More double transformations were done using the competent BL21(DE3) from NEB. We proceeded with BL21(DE3).pU03.pU07 and BL21(DE3).pU01.pU06. Since we figured out pU06 was wrong, the latter transformant was discarded. Transformation was done following the protocol plasmid transformation in E. coli, using 2 μL of each plasmid with a concentration of 2 ng/μL, and a heat shock of 50 s. Here, no colonies were obtained.

We began fluorescent measurement on the strains we had constructed this far: BL21(DE3).pU01, BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, and BL21(DE3).pU01.pU10. OD from overnight cultures was low, so instead of making a new inoculation in the morning, we induced the overnight cultures directly according to the schemes below.

BL21(DE3).pU01.pU07 + arabinose (0.1 % (w/v)), varying IPTG (mM)

BL21(DE3).pU01.pU07 + 1 mM IPTG (mM), varying L-arabinose (% (w/v))

BL21(DE3).pU01.pU08 + manganese (μM)

BL21(DE3).U01.U09 + theophylline (mM)

BL21(DE3).U01.U10 + theophylline (mM)

Measurements after 5 hours were done on a plate reader BioTek Cytation 5.

Week 12

Colony PCR of BL21(DE3).pU03.pU07 was conducted. Correctly looking plasmids were purified and their concentration and purity were checked on a Nanodrop. Additionally, cryostocks were made.

We prepared overnight cultures of BL21(DE3).pU03.pU07 to be used the following week in a fluorescent measurement.

Week 13

Fluorescent measurements were done on BL21(DE3).pU03.pU07. We did the same measurement twice this week.

An overnight culture was inoculated to an OD of 0.05 in LB supplemented with ampicillin and chloramphenicol. After 2 hours an OD around 0.3-0.6 was obtained. Induction was done according to the scheme below using the stock solutions 50 g/L L-arabinose and 100 mM IPTG, the second time a stock of 25 g/L L-arabinose was used for the varying arabinose measurements.

Measurements after 5 hours were done on the plate reader BioTek Cytation 5.

Week 14

USER cloned pU11-14 following USER cloning protocol using 5 μL BioBrick DNA and 1 μL USER-ready vector. Cloned mixture was left at 30°C for 20 min, 20°C for 20 min, and 12°C for 10 min. They were then transformed into competent DH5α cells according to the Transformation in E. coli protocol, and plated onto LB supplemented with ampicillin.

The transformants were genotyped with colony PCR using appropriate primers and following X7 PCR. Cells were diluted in 20 μL MQ, and incubated at 80°C for 5 min. The elongation time was set to 30 s, since the expected band length was 300-400 bp. The PCR products were checked on a gel, and all colonies seemed correct. Overnight cultures of two of each plasmid were made using LB supplemented with ampicillin. The plasmids were purified. Concentrations and qualities were checked on a Nanodrop.

We did another fluorescence test with BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10. The same setup as previously was applied. From overnight cultures, the cells were inoculated to OD 0.1, and left to grow for 2 hours in LB supplemented with ampicillin and chloramphenicol. Stock solutions used were 100 mM IPTG, 50 g/L L-arabinose, and 25 g/L L-arabinose.

Using a 100 mM IPTG stock and 50 g/L L-arabinose.

Using a 25 g/L L-arabinose stock.

Week 15

Double transformations pU11-14 into competent BL21(DE3) from NEB with pU01. Following the protocol Transformation in E. coli. Transformants were plated onto LB supplemented with ampicillin and chloramphenicol. They were left to grow overnight at 37°C. First round of transformations gave no colonies, so a new double transformation was conducted. Using 2 μL of 2 ng/μL plasmids, and following the protocol Plasmid transformation of E. coli, but with 50 s heat shock. Plating 200 μL of the transformants onto relevant media, and left overnight at 37°C.

Colonies were obtained for BL21(DE3).pU01.pU11 and BL21(DE3).pU01.pU13, so these were checked with colony PCR using Taq polymerase. All looked good when run on a gel. Overnight cultures of these were prepared.
The double transformations were done again, since not all gave colonies. They were plated onto LB supplemented with ampicillin and chloramphenicol, and left overnight at 37°C.

pU11-14 was sent to sequencing at Eurofins following the instructions given by Eurofins and with appropriate primers. Results showed that the plasmids were as expected, except for an uninspected insert in the ori, but this could be due to our plasmid map not corresponding perfectly to our plasmid.

Fluorescence test of BL21(DE3).pU03.pU07 was attempted again. An overnight culture was inoculated to an OD 0.04, and left to grow for 2 hours. For the induction following stock solutions were used: 50 g/L L-arabinose stock, 100 mM IPTG stock, and 5 μM anhydrotetracycline (aTc).

The induction plate looked as follows:

[columns 1-3]: BL21(DE3).pU03.pU07 + L-arabinose (0.1 % (w/v)), varying IPTG (mM)
[columns 4-6]: BL21(DE3).pU03.pU07 + 0 mM IPTG, varying L-arabinose (% (w/v))
[columns 7-9]: BL21(DE3).pU03.pU07 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying aTc (uM)

After induction, cells were left to grow for 5 hours. After 5 hours OD and fluorescence was measured on the plate reader SpectraMax i3. Growth was insufficient, so the cells were left to grow further overnight at 37°C.

Another fluorescence test was done on the remaining TMS systems. That being, BL21(DE3).pU01.pU08, BL21(DE3).pU01.pU09, BL21(DE3).pU01.pU10, BL21(DE3).pU01.pU11, and BL21(DE3).pU01.pU13.

As controls, the following was used:

• BL21(DE3): background absorption/fluorescence of the cells.
• pU01: mCherry fluorescence in the low copy number plasmid (must be induced with L-arabinose).
• pIDT02: mCherry fluorescence in a high copy number plasmid (must be induced with L-arabinose).

We used the following stock solutions: 20 mM, 2 μM and 200 μM MnCl2 stock (tetrahydrate - 197.91 g/mol), 25 mM theophylline, 50 g/L L-arabinose, and 100 mM IPTG.

The induction plate looked as follows:
[columns 1-3]: BL21(DE3).pU01.pU08 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying Mn (uM)
[columns 4-6]: BL21(DE3).pU01.pU09 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying theophylline (mM)
[columns 7-9]: BL21(DE3).pU01.pU10 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying theophylline (mM)
[columns 10-12]: BL21(DE3).pU01.pU11 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying PFOA (mM)
[row G-H]: BL21(DE3).pU01.pU13 + 1 mM IPTG + L-arabinose (0.1 % (w/v)), varying PFOA (mM)

Controls are (shown in blue):
LB: 200 μL LB
MQ: 200 μL MQ
BL21(DE3): 100 μL MQ + 100 μL BL21(DE3)
pIDT2 cryo: 96 μL MQ + 100 μL pIDT2 from cryostock (showed growth) + 4 μL L-arabinose (0.1% w/v)
pIDT2: 96 μL MQ + 100 μL pIDT2 from (showed no growth) + 4 μL L-arabinose (0.1% w/v)
pU01: 96 μL MQ + 100 μL pU01 (showed growth) + 4 μL L-arabinose (0.1% w/v)

BL21(DE3).pU01.pU08 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying manganese (uM).

BL21(DE3).pU01.pU09 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying theophylline (mM).

BL21(DE3).pU01.pU10 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying theophylline (mM).

BL21(DE3).pU01.pU13 + L-arabinose (0.1 % (w/v)), varying IPTG (mM).

Using a 100 mM IPTG stock and 50 g/L L-arabinose.
BL21(DE3).pU01.pU13 + L-arabinose (0.1 % (w/v)), varying IPTG (mM).

Week 16

This final week of laboratory work we did our final fluorescence test. We tested our constructs with PFOA-sensitive aptamers and our strain BL21(DE3).pU03.pU07. Thus, following overnight cultures were prepared:

• BL21(DE3).pU01.pU11
• BL21(DE3).pU01.pU12
• BL21(DE3).pU01.pU13
• BL21(DE3).pU01.pU14
• BL21(DE3).pU03.pU07

For controls, overnight cultures of pIDT2 and pU02 were also prepared. All overnight cultures were prepared in LB with appropriate antibiotics. That being, kanamycin for pIDT2, chloramphenicol for pU02, and ampicillin and chloramphenicol for the remaining.

The following day, all strains were reinoculated to OD 0.05.
Stock solutions used for the inductions:

• aTc (5 μM)
• MnCl2 (20 mM, 2 μM, 200 μM)(tetrahydrate 197.91 g/mol)
• Theophylline (25 mM)
• L-arabinose (50 g/L)
• IPTG (100 mM)
• PFOA (0.02 mM and 2 mM)

Column 6 contains PFOA dissolved in dPBS, not MQ.

Controls shown in blue are:

• LB: 200 μL LB
• MQ: 200 μL MQ
• BL21(DE3): 100 μL MQ + 100 μL BL21(DE3)
• pIDT2 cryo: 96 μL MQ + 100 μL pIDT2 from cryostock (showed growth) + 4 μL L-arabinose (0.1% w/v)
• pU01: 96 μL MQ + 100 μL pU01 (showed growth) + 4 μL L-arabinose (0.1% w/v)

Using a 100 mM IPTG, 50 g/L L-arabinose, 5 μM aTc (In MQ).
BL21(DE3).pU03.pU07 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying aTc (mM).

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU13 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU11 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

Using a 100 mM IPTG, 50 g/L L-arabinose, (in orange) 2 mM PFOA (in MQ) and (in green) 0.02 mM PFOA (in MQ).
BL21(DE3).pU01.pU12 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying PFOA (mM).

BL21(DE3).pU01.pU014 + 1 mM IPTG, 0.1 % w/v L-arabinose, varying manganese (μM).

100 μL of each were transferred to a see-through microtiterplate and OD600 was measured. OD was not high enough after 5 h, so the plates were set to overnight incubation (13 h) at 37C. The fluorescence was measured the day after.