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From 2011.igem.org

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Informing Design

We consulted numerous experts in various fields to ensure that the design of the AuxIn system respects all relevant social, ethical and legal issues. One module of our system, Gene Guard, is a direct result of brainstorming around the issues involved in the release of genetically modified organisms (GMOs). Although we have only reached the proof of concept stage, we have put a lot of thought into how AuxIn may be implemented as a product and the legal issues that would be involved.

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Problem: Bacteria frequently pass on genetic information through horizontal gene transfer. This can happen in a variety of ways, including transfer or a mobile plasmid into another bacterium (1). As a result, a significant portion of bacterial genomes (12.8% of Escherichia coli genomes) consists of foreign DNA. (2)

Response: Early on in the project, we recognised that containment of genetic information is an issue that has to be taken very seriously. We discussed many different ways in which we could ensure that the DNA we engineer into our bacteria does not spread to other microorganisms or adjacent ecosystems. We consulted our advisors in the synthetic biology centre at Imperial who informed us about already existing "kill switches". Most of these are based on killing bacteria in response to a specific stimulus. However, many of the compounds that cause activation of these switches could not be applied to soil without side effects. In addition, we could never ensure that all bacteria in the soil would be eradicated through application of these compounds. Switches that kill bacteria after a certain number of replication cycles are also not necessarily applicable as the lysed bacteria leave behind genetic information that can be taken up by other soil microorganisms.

We also consulted two ecologists, who are experts in above-below ground interactions and soil microbial ecology. They both advised us that while it may be more obvious to use naturally occurring soil bacteria such as Bacillus subtilis, Escherichia coli is less likely to survive in soil and may ensure better containment. Dr Alexandru Milcu pointed out that this is especially important considering that very high auxin secretion may skew plant populations. While this is not an issue in areas where the ecosystem is already badly affected, spread to other ecosystems, especially via spores, is a big issue. Dr Robert Griffiths

Action:

GM Release

To prevent spread of the auxin-producing plasmid in the environment, we have devised a containment device that will be able to kill other bacteria that take up the plasmid. In addition, we have devised experiments to test the survivability of E. coli in soil to evaluate whether these bacteria would be outcompeted by other soil microorganisms. Soil-dwelling protozoa also play an important role as they have been shown to feed off bacteria.

Soil Experiment

Although E. coli have been shown to be able to survive in soil for significant amounts of time (more than 130 days in the case of the pathogenic strain O175, as shown by Maule, 2000), the ability to survive even in manure sludge varies greatly between different strains (Topp et al., 2006). To investigate the survival of lab strains of E. coli in soil, we set up a separate experiment. This experiment was started by our work experience student Kiran and carried on by us after he left.

We placed soaked small discs of filter paper in GFP expressing E. coli and put these into autoclaved and non-autoclaved soil. The bacteria were incubated for 10 days. Cultures were grown up in medium containing both ampicillin and kanamycin every day to measure the optical density and thus determine how many bacteria had survived in sterile and non-sterile soil for set periods of time.

To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls.

From day 6 onwards, we started plating the bacteria out rather than measuring the OD to check for retainment of the GFP-expressing plasmid. There were no colonies detected on the negative control plates, showing that OD measurements in the negative control were due to soil particles in the media. The colonies grown up from E coli inoculated in soil were brightly fluorescent, confirming the presence of plasmid-carrying E coli in the soil after a long period of time.

Subsequently, we stopped measuring the OD of bacteria and started plating out cultures from sterile and non-sterile media to check for presence of the plasmid. After three weeks, the bacteria were still growing in non-sterile media and expressing GFP. In addition, there were bacteria present on the plates that were resistant to ampicillin and kanamycin and some of them expressed GFP. It is therefore very likely that the GFP-expressing bacteria passed on the GFP-expressing plasmid to other bacteria in their environment.

References:
Maule, A. (2000) Survival of verocytotoxigenic E. coli O175 in soil, water and on surfaces. Symp Ser Soc Appl Microbiol 29:71-78.
Topp, E. et al. (2006) Strain-dependent variability in growth and survival of Escherichia coli in agricultural soil. FEMS Microbiology Ecology 44:303-308.

Containment Device

The containment device is based on a toxin/anti-toxin system taken from the lysis cassette made by the Berkeley 2008 team. This originally involved the secretion of holin along with lysozyme, so that the holin would form pores in the inner membrane and allow the lysozyme to break down the cell wall. This will cause the cell to lyse when the inducible promoter was induced by arabinose.

Also included in this cassette is the gene for antiholin, under the control of a weak, constitutive promoter to prevent any leakage.

Our project takes this a step further. By using the anti-holin's ability to inhibit the activity of holin, we can create a system in which the lytic activity of holin can be negated by the presence of antiholin in certain cells. This means that we can make a plasmid that can kill one cell, but replicate in another.

References:
(1) Ochman, H. et al. (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405, pp. 299-305.
(2) Thomas, C. & Nielsen, K. (2005) Mechanisms of, and Barriers to, Horizontal Gene Transfer between Bacteria. Nature Reviews Microbiology 3, pp. 711-721.