1.The efficiency of competent cell

We reserched the transformation efficiency of E. coli by using the kit sent from igem. Regarding the protocol, we followed the 2018 kit protocol because the it was not on the 2019 page. However, the liquid amount and the seeding amount were all halved because the amount of competent cells to be used had to be reduced.

When we counted the colonies, there are things that do not grow colonies, but two reasons can be considered. The first is that the colonies may grow slowly in the case that the amount of plasmid is too small, but the time spent on the culture for our convenience was not able to take much enough. The second include that other types of colonies have grown unnecessarily for reasons such as Cm of the medium is little bit.

2.Genetic modification experiment

We took three genes, recA, pprM, and pqqE, from D.rad (NBRC ***). We purified each DNA amplified by PCR by gel extraction, applied them into agarose gel from the left in the order of
marker ( OneSTEP Ladder Marker 100 ),
recA,
pprM,
pqqE,
vector,
marker ( OneSTEP Ladder Marker 500 ),
promorter,
and then conducted electrophoresis. The result we got is shown in Figure 0.

Figure0.

First, we introduced recA gene into pSB1C3 using infusion cloning to transform E. coli. At that time, the cells grew, but when we did insert check, no band was confirmed. After that, we did transformation and insert check again and again, but none of the cells showed any band.

3.Tolerance assay

3.1.Overview

In this page we showed the results of tolerance assay.

Escherichia coli DH5α as negative control showed sensitivity against gamma irradiation.

Deinococcus radiodurans as positive control showed resistance against gamma irradiation.

The transformant E. coli harboring D. radiodurans ’s genes would be more resistant than negative control against gamma irradiation (as a future plan).

3.2.Viability against gamma ray

survival rate of E. coli (negative control)

Figure1. The viability of E. coli DH5α against gamma irradiation

E. coli showed a significant decrease in viability between 50 Gy and 100 Gy versus 10 Gy. From this result, we concluded that E. coli is highly sensitive to gamma irradiation when it is irradiated with 50 and more doses of Gy (As for 0 Gy, an irradiated sample was as same as a non-irradiated group, so the survival rate was calculated as always 1).

survival rate of D. radiodurans (positive control)

Figuren2. The viability of D. radiodurans against gamma irradiation

It showed no significant decrease in the survival rate with respect to the irradiation dose of γ rays, and showed a high survival rate of about 0.9 on average at any irradiation dose. From this result, it can be said that D. radiodurans is radiation resistant (at least with up to 100 Gy).

3.3.Future plans

Figure3. The expected results of transformant E. coli owing D. radiodurans ‘s radiation resistant gene

In figure 3, you can see the expected results of our bacteria harboring D. radiodurans ‘s radiation resistant gene (recA, pprM, or pqqE). We expect that our bacteria would be more resistant than negative control (E. coli) and less resistant than positive control (D. radiodurans). And also, we are going to introduce two or more D. radiodurans genes into one cell, and verify WHICH COMBINATION IS THE BEST TO IMPROVE THE RADIATION RESISTANCE OF E. coli. Eventually, we would like to decide the “key genes” for radio-resistance like Yamanaka 4 factors for iPS cells, and introduce them into the genome of E. coli, establishing a new strain.

Team:Botchan Lab Tokyo/Results