Genetically engineered microbes that are physically contained within research labs or industry bioreactors would pose little or no threat if they somehow managed to escape. But the number of potential applications for engineered organisms has expanded, as well as the scale in which these organisms could be used, necessitating a fresh look at methods for their biological containment, or biocontainment.
The ideal engineered organism does its job and disappears. It does not keep proliferating or unintentionally pass its genes to other cells. Although the idea of biocontainment has been around for decades, interest has reignited in the past few years and—motivated by public concern—more innovation is on its way, said synthetic biologist Karmella Haynes of Arizona State University, who also mentors undergraduates and serves as a judge in the International Genetically Engineered Machines (iGEM) competitions.
“As a pre-emptive protective measure, scientists are building kill switches and containment systems but not because they think the [modified organisms] are dangerous. They’re building containment systems in response to public concern, period,” Haynes added.
Research published in the past year is breaking new ground in biocontainment. Two new Nature studies in particular are the first to create bacteria dependent on synthetic nutrients as a biocontainment strategy, which may help quell uncertainty associated with using genetically-modified organisms (GMOs), bacterial or otherwise, in open environments (1,2).
These papers present “very elegant, extreme examples” of one approach for biocontainment: genetically engineered auxotrophy, in which organisms cannot grow without a particular nutrient in order to survive, said bioengineer Tom Ellis of Imperial College London, who was not involved with the research.
“The original intention was not to solve a plant genome issue and really not even to solve an existing microbial GMO issue,” said geneticist George Church of the Harvard Medical School, who spearheaded the work. “It was to anticipate a future microbial issue and nip it in the bud.”
Recoding for biocontainment
A decade’s worth of work has led to this point. Crucially, in 2013, George Church, Farren Isaacs of Yale University, and their colleagues created a strain of Escherichia coli in which all 321 instances of the stop codon UAG—along with the protein that recognizes it and terminates translation—were deleted from the bacterial genome using multiplex automated genome engineering (MAGE) and conjugative assembly genome engineering (CAGE). Even though the genomes had been recoded, the bacteria survived because there are other stop codons and proteins that work to terminate translation.
“We now had a strain where one of the codons was now effectively without meaning,” said postdoctoral researcher Dan Mandell, lead author of Church’s new Nature study. “We could reassign that codon to a new amino acid so that every time the translation machinery comes across the codon it would incorporate a new amino acid.” Indeed, in that study, they introduced a nonstandard amino acid in place of the UAG.
Working independently after Farren Isaacs started his own lab at Yale University, the scientists then used different strategies to make bacteria depend on an exogenously supplied synthetic amino acid.
Church’s group used computational protein design to make essential enzymes incorporate a synthetic amino acid. This required a reconfiguration of the amino acids surrounding the synthetic one, like a jigsaw puzzle piece, so that the proteins could fold and function properly.
The Isaacs group used a combination of computational protein design and evolutionary approaches, converting the UAG from stop to a sense codon that could encode synthetic amino acids. They used MAGE to site specifically introduce UAG back into precise places in essential genes, genetically encoding a synthetic amino acid at those conserved amino acid positions. Without the synthetic amino acid, the essential genes would not be produced, and the cells would not survive despite having plenty of opportunity and time to escape biocontainment, Isaacs said.
“At the end, what was interesting was that we both presented independently done and distinct solutions that led to species that were dependent on synthetic amino acids,” Isaacs said.
Dealing in uncertainties
Bacteria are abundant, proliferating roughly every 20 minutes and swapping genetic material. As such, it will take a combination of mechanisms to prevent bacteria from coming up with new ways to escape. In a study published in Nucleic Acids Research, Issacs’ group described the incorporation of multiple simultaneously acting safeguards into a single organism (4).
Biocontainment strategies don’t always directly address the potential long-term concern of possible genetic pollution. For example, even if the engineered microbes are killed off using genetic safeguards, some of their DNA could be preserved. If this DNA provides some advantage, such as antibiotic resistance, it could get scavenged by other organisms via horizontal gene transfer (HGT).
One way to address this is to make the DNA less desirable, a strategy Ellis used in an ACS Synthetic Biology paper published last year (5). His group is now working to model and measure the chances of horizontal gene transfer, but “HGT is so rare that even getting good measurements in the lab is quite difficult,” he said.
Church’s group is working to boost dependency on their synthetic amino acid by increasing its abundance. The one described in the new study is several orders of magnitude less abundant in the bacteria than the least frequent natural amino acids.
Both Church’s and Isaac’s groups are working to pressure test the cells further, which requires more cells than can be maintained in a typical academic laboratory.
In the new study, “We took it to less than 1 [escape] in a trillion,” Church said, which falls within the National Institutes of Health’s recommended limit of 1 cell per 108 cells. “To take it further requires some innovation in thinking of how you can quantitate escape at that very low level. We have some ideas there that are moving along quickly,” Church added.
Intriguingly, the strains of the genetically recoded organisms generated in the new Nature studies are resistant to viruses because they employ a different genetic language. This could be beneficial: if the organisms were to be used in industry, they could be less susceptible to viral contamination. On the other hand, if they escaped, their resistance could give them a survival advantage over their natural counterparts. Isaacs said they’ve been studying viral resistance in greater detail and have “some very exciting results.”
1. Mandell DJ, Lajoie MJ, Mee MT, Takeuchi R, Kuznetsov G, Norville JE, Gregg CJ, Stoddard BL, Church GM. Biocontainment of genetically modified organisms by synthetic protein design. Nature. 2015 Feb 5;518(7537):55-60.
2. Rovner AJ, Haimovich AD, Katz SR, Li Z, Grome MW, Gassaway BM, Amiram M, Patel JR, Gallagher RR, Rinehart J, Isaacs FJ. Recoded organisms engineered to depend on synthetic amino acids. Nature. 2015 Feb 5;518(7537):89-93.
3. Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FJ. Genomically recoded organisms expand biological functions. Science. 2013 Oct 18;342(6156):357-60.
4. Gallagher RR, Patel JR, Interiano AL, Rovner AJ, Isaacs FJ. Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Res. 2015 Feb 18;43(3):1945-54.
5. Wright O, Delmans M, Stan GB, Ellis T. GeneGuard: A modular plasmid system designed for biosafety. ACS Synth Biol. 2015 Mar 20;4(3):307-16.