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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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GM Release

There are many different biological aspects that need to be taken into account when deliberating release of genetically modified organisms into the environment. A big issue associated with this is containment of genetic information and preventing its spread to other organisms. To prevent spread of our auxin-producing plasmid in the environment, we have devised Gene Guard, a containment device that will actively prevent horizontal gene transfer. In addition, we have chosen Escherichia coli as our chassis to prevent spread to adjacent ecosystems.

Gene Guard design

1. Goal

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). If the DNA we engineered into our bacteria were to be transferred to plant pathogenic bacteria, it may give them a competitive advantage. This could have detrimental consequences and negatively affect plants and the ecosystem. Therefore, we want to prevent spreading of the genetic information we have engineered into our chassis.

2. Action

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.

3. Result

We took a novel approach towards designing a "kill switch" and designed a containment device that is able to contain genetic information in our bacteria at the same time as preventing horizontal gene transfer to other microorganisms. Gene Guard 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 caused 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.

Chassis choice

1. Goal

In chassis choice, we had to consider several aspects. We wanted to choose a chassis that we would be able to transport to arid areas, preferably already enveloped inside a solid seed coat. In addition, we want the bacteria to be able to persist in the soil long enough to carry out their function. On the other hand, we also want to prevent spread of the bacteria into far-away ecosystems where they are more likely to have a detrimental effect on the ecological balance.

2. Action

We 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 also advised us that while engineering naturally occurring soil bacteria might lead to better persistence and cause our project to be more efficient, containment would be more easily achieved by using bacteria that do not normally occur in soil such as E. coli as they are more likely to be outcompeted.

These arguments caused us to pin-point our chassis choice on B. subtilis, a natural spore-forming bacterium that naturally occurs in soil and E. coli. We initially codon-optimised our genes for both of these species. At the first human practices panel, we thoroughly discussed the advantages and disadvantages associated with both chassis choices (Figure 1).

Figure 1. Advantages and disadvantages of possible AuxIn chassis

3. Result

Containment and possible contamination of other areas is a very big human practices issue. With B. subtilis as our chassis we would never be able to ensure complete containment. On the other hand, enveloping E. coli in a seed coat is mostly a mechanical issue that we should be able to overcome. We therefore chose to use E coli as the chassis for AuxIn.

Survivability of our chassis

1. Goal

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 (3)), the ability to survive even in manure sludge varies greatly between different strains (4).

2. Action

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 over six weeks. Cultures were grown up in medium containing both ampicillin and kanamycin every week. These cultures were plated out and checked for fluorescence to check for presence of the plasmid. To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls.

3. Result

After five weeks, the bacteria were still growing in non-sterile media and expressing GFP. In addition, there were bacteria showing different colony morphologies 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. After six weeks, fluorescence could no longer be detected on the plates grown up from non-sterile soil cultures. Plasmid DNA was extracted by miniprep and digested with EcoRI and PstI to check for presence of the insert. A separate EcoRI only digest was run to check for the size of the opened vector. The colonies from sterile and non-sterile soil both have the colony morphology of E. coli. Nevertheless, 16S ribosomal RNA is about to be amplified by PCR and sent off for sequencing to determine bacterial species.

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