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From 2011.igem.org

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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.

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Testing

Escherichia coli survivability and plasmid retainment in soil

To test for the necessity of Gene Guard when inoculating E. coli chassis into the soil, we set up a soil experiment. We initially transformed chemically competent DH5alpha cells with superfolder GFP. These cells were inoculated on small (about 0.5 cm diameter) filter discs, 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 both sterilised and non-sterilised soil (Fig. 1).

Figure 1. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics imaged using a LAS-3000 gel imager. a) Sample taken from sterilised soil b) Sample taken from non-sterilised soil c) control of non-inoculated filter disc placed in non-sterilised soil. Orange areas indicate areas of green fluorescence (Data by ICL iGEM 2011).

As is visible from these plates, fluorescence was present in bacteria recovered from both non-sterile and sterile soil. The control plate shows 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 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. 2).

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

The insert is very clearly visible at just above 2kb. This confirms the presence of superfolder GFP in both cultures. To check for mutations in the coding sequences of the plasmid, both minipreps have been sent for sequencing.

In addition, small colonies appeared on the non-sterile plate that had very different colony morphology. They were too small to be visible on the LAS-3000 image but appeared to express green fluorescent when visualised with blue light. We grew this colony up in LB medium containing selective antibiotic. Subsequently, we performed a separate miniprep and gel digestion with this colony.

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

References:

(1)