In iGEM 2022, TokyoTech attempted to establish infection detection cell lines by two methods: Electroporation and Lipofection. Infection detecting cells were co-transfected with linearized pCAGGS N-Cre DNA:BBa_K4419025 and linearized pMC1 neo-polyA (Fig.1). The selection with G418 was planned for the gene Neo^R from pMC1 neo-polyA, but transformants could not be obtained because the concentration of G418 was too high. Therefore, we planned an experiment to determine the optimal G418 concentration to obtain transformants.

To determine the optimal G418 concentration to obtain transformants, PULSE was added to Vero cells in the Electroporator and cultured for 1 week in medium graded for G418 concentration. (Experiments 221123~221207) The results showed that G418 900 µg/mL with 30% Confluency after 1 week was optimal for screening (Fig.2).

Electroporation of pCAGGS N-Cre and pMC1 neo-polyA (Both are straightened) with G418 900 µg/mL was performed again as determined in Build and selection with G418. (Experiments 221213-221225) Selections were made for 13 days, but cultured cells did not form colonies and no fluorescence was observed (Fig.3).

We learned from this Test that the concentration of G418 is not the only thing inhibiting the selection of transformants. We also discovered that the resistance gene may not work or the DNA may not enter the cells.

In Cycle 1, we tried to obtain transformants by changing the concentration of G418 but failed. They also found that the resistance gene may not have worked, or the DNA may not have entered the cell. Therefore, we decided to reduce the difficulty of transfection by changing the method to transfection of one type of DNA instead of co-transfection of pCAGGS N-Cre and pMC1 neo-polyA (both are linearized).

This means that Parts BBa_K4419025 has been improved: its promoter has been changed from a CAG promoter to a CMV promoter and a neomycin/kanamycin resistance gene has been added to the gene circuit. BBa_K4828018 was added to the Parts Registry.

As stated in the Design, in order to use a single DNA transfection method, we considered that the Neo-resistant gene should be present in the plasmid backbone that incorporates the genetic circuit of the infection-detecting cell. Therefore, we used pCMV N-Cre, a product in the process of making pCAGGS N-Cre. pCMV N-Cre has pIRES2-EGFP as its backbone and has Neo^R, so only one type of DNA is needed for transformation (Fig.4) .

We electroporated Linearized pCMV N-Cre into Vero as described in Build and performed selection with G418 900 µg/mL as required in Cycle 1. (Experiments 230221-230306) However, no transformants were obtained (Fig.5).

In Cycle 2, the plasmid backbone was changed to reduce the difficulty of transformation, but no transformants were obtained. However, we thought that Vero cells might contain little DNA, so we decided to review the conditions of Electroporation.

As described in Experiments 230412, 230427, Linearized pCMV N-Cre under various PULSE conditions were electroporated and selected with G418 900 µg/mL. (Experiments230412-230417, 230427-230430)

We also stained some of the cells to which PULSE was added with trypan blue to see how many cells were killed by PULSE.

The selection did not result in transformants. In addition, a few cells were stained with trypan blue immediately after PULSE was added.

We were able to confirm by this test that PULSE did not work at all against Vero cells. We thought that this was due to the conditions of the Electroporator or Electroporation.

In Cycle 3 we discovered that there was some problem with our Electroporation. Therefore, we planned to change our transformation method. First of all, we considered changing the conditions of electroporation. However, we soon realized that it was not realistic to search for the optimal PULSE because there were too many conditions to consider. Next, we considered trying Lipofection. However, we gave up because of the high cost of the reagents. So we decided to consult Dr. Suzuki, who has been helping us with SRIP production experiments.

After consulting with Dr. Suzuki, he recommended using PEI: polyethylenimine, which is inexpensive and often used for transfection, and gave us some PEI (Fig.6). (See Human Practices for details.) We immediately performed transfection using PEI. (Experiments 230901, 230905)

The high fluorescence intensity, which was not seen in Electroporation, was confirmed. However, the PEI-based method transformed cyclic pCMV N-Cre instead of linearized pCMV N-Cre (Fig.7).

PEI can successfully transform the cells, indirectly proving that there is a problem with the electroporation conditions as per Learn in Cycle 3. Although PEI involves introducing a circular plasmid, it is still necessary to aim for random integration into the cell genome by transforming linear DNA to isolate transformants, i.e., to establish cell lines.

As described in Experiments230206, we cut out the nluc gene using restriction enzymes and ligated the C-Cre gene as an insert. However, when we transformed the ligation product, no colonies grew. Since we were concerned about the low amount of vector during the experiment, we decided to extract more plasmids from E. coli because we thought it was necessary to prepare a larger amount of plasmids.

Since pCMV YF-C-Cre-rep is a low-copy plasmid, we added chloramphenicol to E. coli culture medium in the logarithmic growth phase. This resulted in a 3-fold increase in the amount of plasmid compared to normal (Fig.8).(Experiments230519)

A sufficient amount of plasmid was digested with SnaB I, the next step. However, there was still some residue, so another overnight restriction enzyme treatment was performed. However, there were still remnants (Fig. 9).(Experiments230607)

From the electrophoresis results of Test, we assumed that there was some reason why the plasmid could not be digested by restriction enzymes.

We found that the plasmid used in Cycle 1 was not digestible by the restriction enzyme. In response to this, we consulted with our PI and considered the possibility that the prolonged incubation of E. coli may have altered the properties of the plasmid.

To achieve what we discussed in Design, we performed plasmid extraction by reducing the incubation time of mass culture of E. coli transformed with pCMV YF-nluc-rep to less than 12 hours.

The plasmid extracted with Build was treated with restriction enzyme with SnaB I, but the remainder of the cut was still visible (Fig. 10).(Experiments230719)

It was found that prolonged incubation was not the direct cause of the inability to process with the limiting enzyme.

In Cycle 1, we found that the plasmid used could not be digested by the restriction enzyme. In response to this, we consulted with our advisor and thought that the restriction enzyme might not be working well because the plasmid was taking on a higher-order structure. Therefore, we decided to perform heat shock to resolve the higher‐order structure of the plasmid.

pCMV YF-nluc-rep (R: SnaB I) was incubated at 65°C for 5 min and cooled rapidly in an attempt to resolve the higher-order structure. Restriction enzyme treatment was later performed again with SnaB I.

Higher-order structures are not the direct cause of the breakage remnant.

Cycle 2-1 and 2-2 were not able to eliminate the remaining pieces. Therefore, we planned to purify only the restriction enzyme-digested portions and use them for downstream.

The completely broken DNA was purified and used in a later ligation reaction for transformation.(Experiments230822-230824)

Colonies grew, so small cultures and small preparations of plasmid were made for all colonies. The extracted plasmids were checked for per colony by restriction enzyme Xma I digestion, but there were none (Fig.12).(Experiments230825-230901)

We had considered plasmid construction using restriction enzymes and ligase for Cycle 1-4, but considering the time remaining, we decided that we would be closer to completion if we tried a different approach.

Consider plasmid constructions without restriction enzymes and ligases.

Inverse PCR was used to amplify all but the nluc gene of pCMV YF-nluc-rep, and the C-Cre gene was incorporated by TaKaRa's InFusion to produce pCMV YF-C-Cre-rep, which was transformed into E.coli.(Experiments230906-230921)

Colonies were grown and colony PCR was performed. The colonies per colony were supposed to show a band at 383 b. However, all the bands that were seen appeared at 563 b or higher. However, the bands that were seen were all at 563 b or higher. Considering that the primers used in this colony PCR annealed to pCMV YF-nluc-rep and produced a DNA fragment of 599 b, it is likely that all the colonies that emerged this time had the original pCMV YF-nluc-rep vector (Fig. 13).(Experiments230922)

We were unable to complete the plasmid construction in InFusion because the PCR products were not purified by the method of cutting them out of the gel, which we believe resulted in contamination of the large source plasmid. Therefore, we believe that the method of gel extraction of the PCR product would allow us to successfully complete the plasmid construction in InFusion.