Although we wish we had started our experiments earlier, we had several troubles regarding the iGEM distribution kit which has not arrived yet. We did the best we could with the iGEM 2021 kit, which was completely degraded. We are positive that the problem was not storage or manipulation though.

Firstly, in order to increase the amount of DNA samples that we had, we did the Polymerase Chain Reaction with previously hydrated dried solutions of our genes: the phospholipase A2 (PLA2) from jararaca, the γPhospholipase inhibitor (γPLI) from jararaca. We also ordered 2 additional circuits of γPLI as a safety measure. For context, those genes are from local serpents and we intend to test those enzyme kinetics against the inhibitors to validate our study.

To estimate the ideal temperatures for the PCR reaction, we used the oligoanalyzer software, then we did some variations to find the ideal temperatures, which were close due to the similar length and structure.

To analyze the PCR, we ran an electrophoresis assay to confirm whether the samples were amplified or not, turns out it did not.

We did the PCR and the electroforesis again, with different temperatures and finally found the ideal temperature for each gene:

γPLI J1: 62 to 64°C

PLA2 J: 62 to 66°C

PLA2 C: 62 to 64°C

Then, we took the PCR product and purified it through a PCR clean up system kit.

With the genes in hands, we started the 3A Assembly protocol, specifically the digestion of our genes with a master mix containing EcoRI and PstI (We chose those enzymes based on our circuit structure).

We chose 3 plasmid backbones candidates to insert our genes: PSB1C3, PSB1A3 and PSB1K3, which are resistant to chloramphenicol, ampicillin and kanamycin, respectively. To guarantee we would not run out of plasmids, we transformed those samples into E. coli Turbo through the heat shock method (30 min submersed in ice, 40 seconds on 42°C and 2 min submerged in ice again). Once the bacteria was incubated (200rpm, 37°C for about 1h), we inoculated the cells into petri plates containing the right antibiotic with LB media. We lay the plates to grow overnight.

Unfortunately, we could not observe any colonies, so we repeated the experiment, but this time we centrifuged the incubated cells to increase the concentration.

After the experiment went wrong again, we decided to confirm whether the plasmid backbones were intact, so we did the electrophoresis assay (70V for 20 min). As we suspected, the backbones were degraded. We chose other wells to perform the experiment again and once again found that the plasmids were degraded. Unfortunately, we observed that all plasmids provided by the iGEM 2021 kit were degraded, and since our iGEM 2022 kit had not arrived, we choose to use the PSB1C3 plasmid provided by the lab itself to continue the experiments.

Finally, we manage to get an intact backbone of PSB1C3, we could observe some metal deformations on the aluminum sheet lf the iGEM kit, it may suggest it is a sign of degradation. Once we confirmed that the backbone was not degraded, we proceeded to the genetic transformation through heat shock, the same conditions as before.

After observing the typical red colonies brought with the RFP (red fluorescent protein) reporter gene, we took some colonies and put them in liquid media to grow overnight at 200rpm and 37°C.

We took the falcon tubes in which we incubated the cells and purified them to remove the plasmids, then we did the electrophoresis to confirm the presence of our plasmid. Then, we quantified the concentration of the plasmid through the nanodrop device, followed by the digestion segment of the 3A assembly protocol. We had to dilute the backbone, for we had a great concentration of plasmids (more than 300 ng/uL).

We digested the genes with the same restriction enzymes (EcoRI and PstI) and ligated following the 3A assembly protocol. Following that action, we did the genetic transformation and layed it to grow overnight.

We could observe several colonies, which were picked with the tip of the pipette to do the colony PCR at 62°C, a temperature that can amplify all of our genes. No genes could be found in the assay.

We did the colony PCR again to make sure that there was not any positive colony and then we did the conventional PCR to increase the amount of genes that we had. The colony PCR went wrong again. The PCR product was purified and quantified (we got values around 40 ng/uL).

Wondering what the problem was, we came to the conclusion that using a master mix with two distinct enzymes at the same time made the effectiveness of the digestion lower than ideal, for the distance between restriction sites is short. Running the reaction with EcoRI and then PstI (the order does not matter really) did enhance the efficiency of the method. We also have purified the digestion product between steps.

We transformed competent cells with our circuit.

Unfortunately, no colony has grown. We kept trying to digest our genes, still unsuccessfully.

We attempted to digest our genes using a protocol from a third party company and ligate through the Gibson Assembly (this is the most used method to ligate parts in our lab). Once everything was on its way, we transformed into E. coli Turbo.

We could observe the formation of colonies. The colony PCR did not show us any of our target genes. We retried the assembly protocol for both the 3A and Gibson. Rinsed and repeated.

As we had many failed attempts and all the workers from the lab did not know what else to try, we decided to switch the T4 Ligase for a new one that has just arrived. Once again it did not work. Given the time we had left, we decided to prioritize our 2 main proteins, the γPLI from jararaca and the Phospholipase A2 from jararaca. Rinsed and repeated.

Finally, we got positive results in the majority of the picked colonies. We proceed to inoculate our positive colonies in liquid media overnight for them to grow.

Once we had our bacteria, we stored some flasks as a safety precaution, and then we purified following a purification kit. We transformed into E. coli BL21 DE3 Rosetta, but it did not work out because it has the same antibiotic resistance as our circuit. Then, we started working with Arctic Express E. coli BL21 DE3.

We prepared competent cells for genetic transformation and transformed through heat shock, we laid the petri plates containing our transformed cells overnight.

Today we did the colony PCR with the transformed Arctic Express E. coli to check if we had a positive colony (25 uL of sample). After this, we did the electroforesis of the samples that did not show positive results. Thus, tomorrow we will probably make plasmid digestion and the ligation of parts again, but with another plasmid, the PSB1A3, and put this into E. coli turbo.

We did today colony PCR with the Arctic express E. coli and Turbo E. coli, both of them had parts 𝛾PLI jararaca and PLA2 jararaca into the PSB1C3 plasmid. The turbo E. coli we couldn't do the PCR because the colonies were very close to each other, forming a “carpet”. So, we did this experiment only for the Arctic express and we have the positive colonies! After this, we put the inoculum of positive Artic express colonies to grow in the LB cultivation medium overnight.

Today, we used the tubes containing E. coli and LB medium to be the inoculum, the control and the expression, following our chassis protocol. We did the stock cells (glycerol + 500 uL sample) and storaged it into the ultrafreezer.

For the control medium, we used 60 uL of the sample + LB without antibiotics and we incubated for 3 hours.

Then, we used 100 uL of inoculum after the incubation and put this at 10 degrees Celcius into the shaker overnight. Then we added IPTG to induce the production of proteins.

SDS-PAGE gel preparation: water +SDS +acrylamide + ammonium persulfate + TEMED + tris HCl

We took the control sample and left it growing this overnight to run into acrylamide gel.