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 sterilised soil. Colonies appearing to be on the plates from non-sterile soil had lost fluorescence (Fig. 2).

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).

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).

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)

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.

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