The use of genetically modified organisms (GMOs) is a highly valuable tool in the Scientists arsenal. However, it does not come without its limitations, such is the issue surrounding biocontainment. Three potential ways in which organisms may overcome biocontainment are:
-Mutagenic drift, in which the organism may spontaneously mutate, losing the toxin kill switch.
-Environmental supplementation, for example engineered auxotrophy, the environment is able to provide the amino acid which they cannot survive without
-Horizontal gene transfer when genes are transferred from one organism to another. In this way they are able to then enter the environment.

These are all considerations that we have contemplated as a result of our project. We have been in contact with Dr Farren Isaacs, one of the authors of the studies that we had been investigating (Rovner et al, 2015). The biocontainment issue is so elegantly dealt with by engineering a synthetic amino acid using a redundant stop codon. Also the integration of the converted codon sequence directly into the E.coli chromosome appears to ensure the genes will be continuously replicated without fear of rejection from the cell. This method has not as of yet been used, to our knowledge, as a proven biocontainment technique. This may partially be due to the financial restraints. The budget of the Westminster iGEM team did not cover the cost of such a method. In our communication he directed us to the Gallagher et al, 2015 study.

The novel techniques employed by Gallagher et al (2015) were based on developing a number of ‘safeguards’ such as synthetic riboregulators, responsible for controlling expression of essential genes, and an elegant kill switch that is activated if the organism leaves the media from which it is growing in order to prevent escape. The kill switch was in the form of nucleases that in effect cleaves the host (Escherichia coli) genome. Overall the effect is to ensure that the organism cannot escape the media from which it is growing as the media itself also contains molecules vital to the operation of the riboregulators and, hence, survival of the cell. This could be a consideration for our project. However we did not further explore these techniques due to time and financial constraints.

Another option is to create a double plasmid interdependent toxin-antitoxin system. Each plasmid will contain a toxin and an antitoxin where the toxin will find its corresponding antitoxin on the other plasmid. This ensures the bacteria require both plasmids to survive. The likelihood of both plasmids being transferred to native bacteria is unlikely (Hayes, 2003). This may still encounter issues regarding horizontal gene transfer and mutagenic drift. However, the use of kill switches is still a consideration.

The use of the redundancy of codon usage is one such technique (Mandell et al, 2015). There are numerous codon encoding just one amino acid, for example leucine which has the possibility of 6 different codons. Therefore, by utilizing this redundancy, Mandell et al (2011) were able to mutate each TAA to read for one of the other two stop codons. In doing so releasing TAA to be used as a ‘free’ codon, for example to introduce a synthetic amino acid. Although this appears such a simple idea, there are many other factors and other machinery involved, not just the codons, the recognition machinery such as tRNAs and the amino acids themselves.

We are very excited by our project and want to cover all aspects to ensure its safety but also success. Biocontainment is an issue that needs to be addressed in order to viably use GMOs. Although the techniques above may be expensive, as with many new techniques, such as PCR and genome sequencing when they first emerged, it is still important to make serious considerations into these novel methods in order to make it more financially possible. We, 2015 Westminster iGEM team, believe that biocontainment is one of the key issues surrounding synthetic biology. It is paramount in order to ensure that the GMO of interest does not escape into the environment. The use of TAA stop codon and the use of synthetic amino acids are elegant techniques. Although time consuming within the time and financial constraints of the iGEM competition, in the future and for future work we would like to pursue the potential of introducing such techniques as they appear impervious to horizontal gene transfer.

Key References
Gallagher, R, R., Patel, J, R., Interiano, A, L., Rovner, A, J., and Isaacs, F, J. (2015). Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Research 1-9

Hayes, F., (2003). Toxins-Antitoxins: Plasmid Maintenance, Programmed Cell Death, and Cell Cycle Arrest. Science, 301(5639), pp.1496-1499.

Mandell, D, J., Lajoie, M, J., Mee, M, T., Takeuchi, R., Kuznetsov, G., Norville J, E., Gregg, C, J., Stoddard, B, L., and Church, G, M. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature 518: 55-74

Rovner, A, J., Haimovich, A, D., Katz, S, R., Li, Z., Grome, M, W., Gassaway, B, M., Amiram, M., Patel, J, R., Gallagher, R, R., Rinehart, J., and Isaacs, F, J. (2015). Recorded Organisms engineered to depend on synthetic amino acids. Nature 518: 89-105