1.2 Plasmid retainment

After observing survivability and growth of E. coli in soil, we assumed that the plasmid containing sfGFP (BBa_K515105) had been retained in the cells for 7 weeks either due to the bacteria being in static state or due to very slow growth of the cells. In order to test this assumption, we wanted to observe the retainment of plasmid within our chassis in optimal growth conditions without the presence of selective antibiotics. For this observation we required cells which would be continuously dividing. We grew cells in LB for a number of days and supplied the bacteria with fresh LB medium each day so that the microbes went through the different growth phases of bacterial maturation every day. In addition, a small proportion of the culture was plated on an antibiotic-containing agar plate each day and grown overnight to observe the extent of plasmid loss.

Figure 3: E. coli cells grown in LB without antibiotic and subsequently plated on the selective plate have been imaged using Fujifilm LAS 3000 Imager. The samples have been plated after a) 1 day, b) 14 days. In sample after 1 day growth single colonies can not be observed due to high cell density which was plated. In sample after 14 days of growth, decrease in the bacterial density and decrease in fluorescence can be seen. The bacteria were imaged using GFP excitation and emission wavelengths and the picture was kept in the original greyscale format. (Data by Imperial College iGEM team 2011).

We could observe that even after fourteen days of continuous growth in optimal conditions without the presence of antibiotics, our chassis still managed to express sfGFP and therefore contained the plasmid. However, considerable loss of fluorescence can be seen due to progressive loss of plasmid. Also, a decrease in the number of colonies can be observed. The lower number of colonies is due plasmid loss. Plasmid loss can be attributed to the replication rate of the bacteria being faster than the replication rate of the plasmid.

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2.2 Gene integration of the anti-holin

Once we knew that the anti-holin construct was alright and seemed to be producing a small protein we had to excise the insert and ligate it into the CRIM plasmid. This was complicated as the high copy number plasmid was placing a heavy metabolic burden on the cells making them grow incredibly slowly (something that is not desirable for when you have to be working for a deadline). Not only that, the cells seemed to be losing the plasmid after four days on a plate in a cold room and some of the colonies managed to alter the plasmid in a way that made it lose the sfGFP and allowed the cells to grow quickly and out-compete any cells with the correct plasmid. However, after a lot of trial and error we managed to isolate eight plasmids that were possible candidates for the plasmid we wanted to integrate.

Figure 5: Gel of colonies suspected of containing anti-holin in the genome. The amplified bands of all the colonies correspond to around 300 bp which is approximately the size of the anti-holin gene. The lane furthest to the right is a control in which the anti-holin gene is contained in a pSB1C3 plasmid. (Data by Imperial College London iGEM team 2011).

In order to test these eight colonies we first did an EcoRI and PstI digest and then looked at the size of the insert compared to a control which only contained sfGFP in the CRIM. According to our results, three of the colonies contained an insert that was larger than that of the control. However, the gel red we used to stain the gel seems to do something funny with the buffer we were using to digest making all the bands run higher than they should be running. Therefore, to conclude that the plasmid contained the inserts we wanted we looked for a restriction site that is unique on the anti-holin and the CRIM. We found that the anti-holin construct contains a ClaI site that is also present on the CRIM vector. We performed this digest in another buffer and obtained the expected bands for the three colonies confirming that they contain the anti-holin on the CRIM.

Then we performed the genome integration step which involves the transformation of a cell line containing the helper plasmid^[2]. In order to test whether the colonies had integrated the CRIM in the correct location we ordered the primers that were used in the original CRIM paper and performed a colony PCR on all the colonies. A few of the colonies had integrated the CRIM twice which could be clearly seen under a blue box. However, the rest of the colonies had a single integration event which could also be seen by their phenotype under blue light.

Finally, we had to prove that the anti-holin gene was now present within the colonies. In order to achieve this we performed numerous PCR's with all kinds of buffers and polymerases until we finally got one to work. Using Taq polymerase and Barnes' buffer we managed to amplify a band of the appropriate size using the same primers that we used to initially amplify the anti-holin from its origin.

As of wiki freeze, we are currently having issues with curing the cells of the helper plasmid. We hope to have this fixed and finally test the system before we fly off to Boston. For the conclusion of this module, please come and see our presentation at MIT.

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