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Team:Imperial College London/Project Gene Testing
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<h2><i>Escherichia coli</i> survivability and plasmid retainment in soil</i></h2> | <h2><i>Escherichia coli</i> survivability and plasmid retainment in soil</i></h2> | ||
<p>To test for the necessity of Gene Guard when inoculating <i>E. coli</i> 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.</p> | <p>To test for the necessity of Gene Guard when inoculating <i>E. coli</i> 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.</p> | ||
| - | <p>After six weeks, we were able to recover fluorescent bacteria from both sterilised and non-sterilised soil. | + | <p>After six weeks, we were able to recover fluorescent bacteria from both sterilised and non-sterilised soil (Fig. 1). |
| + | <p>INSERT IMAGE</p> | ||
| + | <p><i>Figure 1. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics. 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.</i></p> | ||
| + | <p>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 gel digest to check for presence of the original insert. | ||
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Revision as of 14:53, 19 September 2011
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
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).
INSERT IMAGE
Figure 1. Colonies recovered from filter discs and grown on LB plates containing selective antibiotics. 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.
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 gel digest to check for presence of the original insert.
