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Proposed Implementation

Lab Notebook

Here follows a weekly summary of our notebook, briefly describing the work we did and what we accomplished each week. For more information about the protocols we followed and the experiments performed, see Experiments.

Week 1 (June 15-19)

LB medium, an ampicillin stock and petri dishes with solid LB medium were prepared, with and without ampicillin. LB medium, pipette tips, eppendorf tubes, glycerol, CaCl2-solution and RNAse free water were autoclaved. E. coli TG1, TG1 (pET-11a), and BL21 (DE3) were plated, incubated and stored in a cold room.

Week 2 (June 22-26)

Our supervisor introduced us to the department's centrifuges, spectrophotometers and plasmid purification kit. Competent TG1 cells were prepared and stored at -80°C. TG1 (pET-11a) cells were incubated overnight, the plasmids were purified and DNA concentration was measured using a spectrophotometer. A test digestion with restriction enzymes NdeI and BamHI was performed to test appropriate reaction times for each enzyme. Digested plasmids were dephosphorylated with FastAP and loading dye was then added.

Week 3 (June 29- July 3)

A gel electrophoresis was performed with the plasmid samples from the previous week. NdeI seemed to not have digested the plasmid fully, while BamHI had started to digest the plasmid unspecifically, due to too long reaction time. New test digestions were performed with longer reaction times for NdeI (samples taken at 60-130 min) and shorter for BamHI (samples taken at 6-15 min). After examining the gel of another test it was decided to run the reactions for 60 min with NdeI and 5-10 min with BamHI.

Week 4 (July 6-10)

Double-digestion and dephosphorylation of the purified pET-11a was done, then a gel electrophoresis was performed with the samples, showing that the digestion was complete. A gel extraction was performed and the digested plasmid was eluted in an elution buffer, then stored at -20°C. E. coli BL21(DE3) was incubated overnight and prepared to make competent cells, then stored at -80°C.

Week 5 (July 13-17)

We had finished all the preparations that we could do and waited for IDT’s free 20k bp, which were delayed. We then decided we could not wait for the free bps and ordered part of our gBlocks ourselves. We ordered all AMPs with Trx as a fusion partner (Trx-gblocks). GST-Scarabaecin was accidentally ordered instead of Trx-Scarabaecin. Previously ordered primers arrived from IDT.

Week 6 (July 20-24)

A new plasmid purification was performed after an overnight incubation of TG1 (pET-11a) and DNA concentration was measured.

A test amplification of gBlocks was performed using the primers on one of the inserts. The PCR machine’s temperature gradient was used to find the best annealing temperature of the primers. After examining the agarose gel of the PCR product we decided to use an annealing temperature of 66°C. The Trx-gblocks (and GST-Scarabaecin) were amplified using that annealing temperature.

Week 7 (July 27-31)

Amplified Trx-gblocks were double digested with NdeI and BamHI, they were run on a gel and purified, in the final step eluting them in nuclease free water. Concentrations of all the samples were measured. Ligation of pET-11a and Trx-gblocks was performed and the product was transformed into E. coli TG1. Cells were spread on plates with LB medium containing ampicillin. A positive control with TG1 transformed with purified undigested pET-11a and a negative control using TG1 transformed with digested pET-11a without any insert was also made.

After incubation some colonies were present for MsrA2, EAFP1, Ec-AMP-D1 and Temporin A. Colony PCR was performed and run on gel, results were not convincing. Two more ligations and transformations were performed as above, however no colonies were present except for on the positive control. Digestion with NdeI and BamHI was tested again to ensure this was not an error and it did not seem to be a problem.

Week 8 (August 3-7)

A fourth ligation and transformation was performed, this time using more ligase in the ligation mixture and prolonging the ligation time from 30min to >120min. Cells were also incubated with the ligated plasmids for a longer time. Still no colonies were found after incubation on plates.

A new double digestion with NdeI and BamHI was performed for the amplified Trx-gblocks. They were purified from a gel and DNA concentrations were measured. TG1 (pET-11a) was incubated overnight and plasmids were again purified and digested by NdeI and BamHI, DNA concentration was measured.

A fifth ligation and transformation was performed, using the new materials and prolonged ligation and incubation times. This time colonies were present for all different g-blocks, there was no growth on the negative control and plenty of growth on the positive control.

A new set of gBlocks arrived from IDT (Delayed free kbp), which contained all AMPs fused to GST as well as Temporin A and Scarabaecin fused to Trx and also our Enterokinase composite part.

Week 9 (August 10-14)

After a first failed colony PCR, another colony PCR was performed picking five colonies per Trx-insert. All inserts had at least 3 colonies that appeared to have the correctly ligated plasmid. Before the PCR, the colonies were also plated on a masterplate and incubated. From the masterplate, clones of each insert were incubated and their plasmids were purified. Plasmids were sent to Eurofin for sequencing and the received data was analysed, showing that almost all gblocks had been inserted correctly without mutations occurring.

The new set of gblocks (GST-inserts) was amplified and purified.

Week 10 (August 17-21)

The recombinant plasmids were transformed into BL21, TB-medium and IPTG-stock were prepared. Inoculations from master plates were incubated overnight (one per Trx-insert) and protein expression cultivations were started the day after for the recombinants. Cultivations were induced using IPTG and samples were taken during a period of 24h. A test SDS-page was performed to learn the technique.

The GST-inserts were double digested with NdeI and BamHI. The enterokinase part was amplified and digested. The digestion reactions were run on a gel and extracted, eluted in water and DNA concentrations were measured. Transformation was made into TG1 cells and colonies were formed for all inserts except GST-Rs-AFP1. A colony PCR and masterplate was performed, however something went wrong with the reaction.

Week 11 (August 24-28)

Samples from cultivation had been centrifuged and were resuspended to obtain the same OD. SDS-page was performed for the samples but did not give any clear results, it did however seem like the proteins had not been expressed for all samples except one. In a GST-scarabaecin sample there was a thick band not present in controls or other samples.

A second protein expression cultivation was performed according to the same protocol as the previous one. Samples were taken and OD was measured during the 24h period, and at the end of expression all cells were harvested by centrifugation, storing the cell pellets on ice. New SDS-pages were made for samples taken during the expression, but something went wrong with the staining. The harvested cell pellets were resuspended and sonicated, pellets and supernatants were stored separately.

Week 12 (September 1-5)

SDS-page was carried out for the pellets and supernatants of the harvested, sonicated cells. After examining these gels and the gels from the previous week, we had a strong belief that GST-Scarabaecin had been expressed. This was the only insert with GST instead of Trx and we started having suspicions that GST might yield higher expression than Trx.

Week 13 (September 8-13)

Pre-inoculation was prepared for a third protein expression cultivation.

Week 14 (September 14-20)

The third cultivation was started and was performed the same way as the previous tries. The harvested cells were treated in three different ways to try and solubilize potential proteins. The treatments were resuspension in SDS loading dye (as before), resuspension in urea and then SDS loading dye, and lastly resuspension in guanidine hydrochloride and then SDS loading dye. SDS-pages of the supernatant (after sonication) and the three treatments were carried out, however the target proteins did not appear present.

Week 15 (October 5-10)

For the second set of parts, a new colony PCR was performed for the clones of the previously made masterplate. However no convincing results of successful transformation were obtained, and it was decided that we no longer had time to continue the work on these parts.

For the first set, a new protein cultivation was started to try and repeat the expression of GST-Scarabaecin (and possibly others). After making SDS-pages of samples taken at 6h and 8h after IPTG induction results showed that there had been no significant protein expression. We decided that we would have to call an end to the lab work, and try to settle with the results that we had gotten.

Week 16 (October 13-17)

The last SDS-pages were for the protein cultivation done the week before. The SDS-pages finally showed what appeared to be protein expression! Things were cleaned up in the lab.

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