Industrial Safety - Biocontainment
Safe use of genetically engineered machines involves physical containment, safe practices as well as biocontainment methods to prevent escape, survival and proliferation of engineered microorganisms in natural environments. In consideration of industrial scale-up of our light-activated biosynthetic process, we have outlined an innovative strategy towards biocontainment published in Nature earlier this year.
Biocontainment methods create intrinsic biological barriers between the engineered microorganism and the natural environment. Previous strategies include metabolic auxotrophy and conditional suicide toxin/antitoxin switches. However these are readily compromised by supplementation of required metabolites by the environment or other organisms, leaky expression of essential genes and mutational escape (either spontaneous, due to evolutionary pressure or through swapping DNA with wild-type organisms i.e. horizontal gene transfer).
This new approach uses genetically recoded organisms (GROs), which require synthetic building blocks for protein synthesis and thus, survival. These GROs have amber STOP codons (TAG) incorporated in essential genes throughout their genome (with the normal occurrence of TAG replaced with alternative stop codons). Orthogonal translational machinery is able to convert this stop codon into a sense codon and incorporate synthetic amino acids (sAAs) into these essential proteins (while maintaining proper translation, folding and function of these proteins). Therefore the microorganisms cannot grow unless the sAA is exogenously supplied.
These strains have amino-acyl tRNA synthetases that will attach the sAA to a suppressor tRNA synthetase that reads the amber stop codon, allowing the sAA to be incorporated into peptides at this site. They also lack release factor 1 (RF1) as a basis for the GRO, as RF1 mediates translational termination at the amber stop codon (which would hinder incorporation of the sAA).
The success and complexity of this biocontainment system can be increased in a number of ways. Incorporation of the amber stop codon into multiple essential genes means that a single rescue mutation will not compromise the system. Converting multiple stop codons, e.g. amber and ochre stop codons into sense codons for sAAs mean that if one codon starts incorporating a natural amino acid, the other maintains the biological barrier. Furthermore, combinations of essential proteins can be optimised such that if the synthetase begins to incorporate a natural amino acid that is structurally favoured in some essential proteins, it will be disfavoured in others, thus decreasing the overall fitness of the escapee.
One last, very meta, strategy is to incorporate an amber stop codon into the amino-acyl tRNA synthetase specific for the sAA. As the synthetase requires the synthetic amino acid for its expression and function, the synthetase itself is only able to incorporate sAAs into other proteins if the sAAs are present - and thus are less likely to incorporate a natural amino acid in the absence of sAAs.
REFERENCES
Mandell, D. J., Lajoie, M. J., Mee, M. T., Takeuchi, R., Kuznetsov, G., Norville, J. E., Gregg, C. J., Stoddard, B. J. & Church, G. M. Biocontainment of genetically modified organisms by synthetic protein design. Nature. 518, 55-60 (2015).
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. & Isaacs, F. J. Recoded organisms engineered to depend on synthetic amino acids. Nature. 518, 89-93 (2015).
