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Module 3: Gene Guard

Containment is a serious issue concerning the release of genetically modified organisms (GMOs) into the environment. To prevent horizontal gene transfer of the genes we are expressing in our chassis, we have developed a system based on the genes encoding holin, anti-holin and endolysin. We are engineering anti-holin into the genome of our chassis, where it acts as an anti-toxin, and holin and endolysin on plasmid DNA. In the event of horizontal gene transfer with a soil bacterium, holin and endolysin will be transferred without anti-holin, rendering the recipient cell non-viable and effectively containing the Auxin Xpress and Phyto-Route genes in our chassis.

Testing

1. Anti-holin expression

We have completed stage 1 of the assembly of the Gene Guard. A protein gel showed a clear band of the appropriate size right at the bottom of the gel when compared to a control cell. Therefore, we have sequence verified and shown that the BBa_K515104 expresses a protein of the appropriate size (Fig. 1).

2. Escherichia coli survivability and plasmid retainment in soil

Figure 3. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from non-sterilised soil b) Sample taken from sterilised soil (Data by Imperial College London iGEM 2011).

We set up a soil experiment to test how long our E. coli chassis can retain its plasmid in soil. We initially transformed chemically competent E. coliDH5α cells with superfolder GFP. These cells were inoculated on small filter discs (about 0.5 cm diameter), which were placed in autoclaved and non-autoclaved soil. We periodically grew up cultures from these filter discs over the course of six weeks.

After six weeks, we were able to recover fluorescent bacteria from sterilised soil. Colonies appearing to be by morphology on the plates streaked from non-sterile soil had lost fluorescence (Fig. 2).

Figure 2. Gel digests of bacteria displaying colony morphology typical of E. coli recovered from non-sterilised and sterilised soil. These bacteria exhibited colony morphologies typical of E. coli. (Data by Imperial College iGEM 2011)

As is visible from these plates, fluorescence was present in bacteria recovered from both sterile but not from non-sterile soil. The control plate showed that there was no contamination with other fluorescent lab bacteria. In order to investigate whether the fluorescence observed was due to the presence of the original sfGFP construct and whether the E. coli-like colonies from the non-sterile sample had retained a plasmid, we extracted plasmid DNA using a miniprep kit and did a digest with EcoRI and PstI and with EcoRI on its own to check for presence of the original insert and size of the unfolded vector, respectively (Fig. 3).

The insert is very clearly visible at just below 2 kb. This confirms the presence of superfolder GFP in both cultures. Sequencing of the GFP insert revealed that a singl frameshift mutation had taken place in the colonies grown up from non-sterile soil. No mutations were observed in the superfolder GFP gene contained in the bacteria inoculated in non-sterile soil. This explains the absence and presence of fluorescence in the respective colonies. However, the bacteria were still resistant to the antibiotics and contained the plasmid. We will be replicating these results with other samples to ensure this is representative.

In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. We grew this colony up in LB medium containing selective antibiotic and subsequently performed a separate miniprep. No DNA was yielded in this miniprep. It is therefore likely that the plasmid was not transferred to these bacteria but that they either possess natural antibiotic resistance or were able to survive on plates that whose antibiotics had already been depleted by the presence of resistant engineered bacteria.

All three samples, the sample from the sterile plate, and the two from the non-sterile plate showing E. coli-like and non-E. coli-like morphologies will be used for 16S ribosomal RNA sequencing (using commonly used primers ^[1]) to determine the bacterial species.

This result is extremely important as it shows that plasmids can be retained in E. coli for a very long period of time even in the presence of competition when inoculated in soil. This gives us an indication of the life-span our chassis would have in the soil in its implementation stage. In addition, long retainment of the plasmid means that the chance for horizontal gene transfer increases, rendering this result very important for the Gene Guard module.

References

[1] Tanner M et al. (2000) Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedius from a canine pet to a human. Journal of Clinical Microbiology 38(4): 1628-1631.