There is considerable public concern about the use of genetic engineering, particularly around the production of food. The most recent example is the FDA-approved, fast growing salmon from the company AquaBounty. This fast growing fish is grown in land-locked tanks as sterile females so that they cannot reproduce if they were to escape into the wild. Interestingly, this fish grows almost twice as fast as salmon in the wild due to the increased production of a fish growth hormone, genetically engineered to be expressed from multiple organs instead of in limiting amounts from the pituitary gland. The basic research that led to this transgenic fish was described over 20 years ago from a Canadian researcher, Dr Garth Fletcher of Memorial University.
What is less appreciated by the public are the numerous examples of genetic modification that result in medicines or other useful and less controversial products. One of the first products resulting from genetic modification was the production of synthetic insulin used to treat diabetes, which is expressed and purified from bacteria. And the exploding field of synthetic biology has the potential of providing a bounty of new products, medicines, bioremediation strategies using engineered bacteria or biofuel production by microbes. If genetically engineered bacteria are to be employed in the field, at sites of contamination, or in production factories, there is a significant worry about their escape and the possible implications of release into the wild. To address this concern, numerous research groups are designing so-called ‘kill switch’ strategies, which will prevent the growth of these microbes in the environment. A creative new strategy was described earlier this year.
Microbiologists frequently work with ‘auxotrophs’, which are strains that cannot synthesize a specific nutrient, and therefore do not grow without its supply to the microbe. As standard auxotrophs might be capable somehow of scavenging these needed nutrients, ‘synthetic auxotrophs’ were designed that rely on the supply of unusual amino acids that are not commonly found in nature. There are several uncommon amino acids that are coded for in the triplet DNA code, but that are not found very often in proteins. This suggests the possibility to change the genetic code and introduce new codons that would introduce non-standard amino acids into proteins.
The first stage of the kill switch technology was founded on an earlier landmark paper that used whole genome editing, where all 321 UAG stop codons were replaced with UAA codons (ref 1). UAG was convenient to alter, since it appears rarely in the genome and only at the end of genes. These strains are referred to as genetically recoded organisms, or GROs. Note, there were 355 off target mutations created in the process, which did not affect viability but may need to be corrected in future. It should be noted that this GRO strain was now quite resistant to infection from viruses, another advantage to this type of biocontainment strategy. The new genetic code mistranslates viral proteins that hijack the bacterial protein synthesis machinery, which ultimately disables new virus assembly leading to resistance.
Several other sophisticated, genetic manipulations were performed in order to create a growth dependence. It was now possible to reassign UAG as a dedicated sense codon for “plug and play” incorporation of rare amino acids. In order to translate the UAG codon to a meaningful response by the protein synthesis machinery, they expressed a specific tRNA synthetase that recognizes the UAG codon and introduces 1-4,4′-biphenylalanine (bipA) into target proteins during synthesis. As there were no longer any functional UAG codons in the genome, they selected a handful of essential proteins and redesigned these genes to encode UAG at various positions. Structure predictions were used to select the candidate essential genes to recode, which were confirmed by protein structure determinations. Mass spectrometry also confirmed the introduction of bipA into target proteins.
Unfortunately, some synthetic auxotroph strains were still viable at low frequencies when grown in the the absence of bipA, indicating that compensatory mutations occurred which restored viability. In other words, the supposed ‘kill switch’ was not perfect in the earliest attempts. Several additional mutations were needed, including the addition of conventional auxotrophs, to really limit growth and escape of this strain in the absence of the non-standard amino acids. They ultimately identified a highly engineered strain could only survive and produce functional essential proteins when grown in the presence of the non-standard amino acid, 1-4,4′-biphenylalanine (ref 2). This ‘tour de force’ approach created a synthetic auxotroph which now can now be used as the ‘chassis’ for the future development of a particular biotechnology.
In the same issue, a second paper on the same subject was also published. They generally used a similar approach to construct synthetic auxotrophs that were dependent on the exogenous supply of unnatural analogs of phenylalanine (ref 3). The end result was the same, a strain that could not escape its amino acid dependency through mutation or the acquisition of multiple genes through horizontal gene transfer. The NIH standard for an effective kill switch is an approach where survivors do not occur with a frequency of 1 per 108 cells. Other non-auxotroph strategies have been used that meet this minimum requirement. Environmental microbes capable of degrading environmental pollutants have been designed to express a lethal toxin when in the environment, but only once the pollutants have been degraded. This permits the deliberate use of bacteria for bioremediation of contaminated soils but limits their survival after the job is done. These bacterial suicide strategies were developed to mitigate possible risks and should permit the safe use of microbes in the environment to help resolve environmental or health crises, with the benefit greatly outweighing the risk.
Ref 1. Genomically Recoded Organisms Expand Biological Functions. 10.1126/science.1241459
Ref 2. Biocontainment of genetically modified organisms by synthetic protein design. doi:10.1038/nature14121
Ref 3. Recoded organisms engineered to depend on synthetic amino acids. doi:10.1038/nature14095