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• W3026544066 abstract "Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority. Here, we present an in-depth summary of biological containment systems for micro-organisms published to date, including the production of a genetic firewall through genome recoding and physical containment of microbes using auxotrophies, regulation of essential genes, and expression of toxic genes. The level of containment required to consider a transgenic organism suitable for deployment is discussed, as well as standards of practice for developing new containment systems. Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority. Here, we present an in-depth summary of biological containment systems for micro-organisms published to date, including the production of a genetic firewall through genome recoding and physical containment of microbes using auxotrophies, regulation of essential genes, and expression of toxic genes. The level of containment required to consider a transgenic organism suitable for deployment is discussed, as well as standards of practice for developing new containment systems. Microorganisms have been used inadvertently in the processes behind food production for thousands of years, with various bacterial and fungal species playing key roles in brewing beer and the fermenting of wine, the baking of bread, and the process of turning milk to yogurt. However, microbiology as a scientific discipline did not solidify until the 17th century, with the exploration of microorganisms as the possible vector for human and animal diseases. It was with these observations that the motivation to control and contain bacterial growth became apparent. Medical advances attributed to sterile and abiotic techniques played an integral part in the control of pathogens. In the 20th century, the discovery and use of antibiotics lead to a revolution in bacterial containment by the application of small molecules. Today we use microbes for an astonishing range of functions. In addition to the aforementioned production of ethanol, CO2, and lactic acid for alcoholic drinks, bread, and yogurt, respectively, both wild-type and engineered microbial species are used to produce a variety of industrially relevant products. Examples include enzymes such as amylase (Sundarram and Murthy, 2014Sundarram A. Murthy T.P.K. α-Amylase Production and Applications: A Review.J. Appl. Environ. Microbiol. 2014; 2: 166-175Google Scholar, Yoneda, 1980Yoneda Y. Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation.Appl. Environ. Microbiol. 1980; 39: 274-276Crossref Google Scholar) and proteases (Razzaq et al., 2019Razzaq A. Shamsi S. Ali A. Ali Q. Sajjad M. Malik A. Ashraf M. Microbial proteases applications.Front. Bioeng. Biotechnol. 2019; 7: 110Crossref Scopus (198) Google Scholar); polysaccharides for use in food, cosmetics, and pharmaceutical products such as xanthan gum (García-Ochoa et al., 2000García-Ochoa F. Santos V.E. Casas J.A. Gómez E. Xanthan gum: production, recovery, and properties.Biotechnol. Adv. 2000; 18: 549-579Crossref PubMed Scopus (1011) Google Scholar) and dextran (Sarwat et al., 2008Sarwat F. Ul Qader S.A. Aman A. Ahmed N. Production and characterization of a unique dextran from an indigenous Leuconostoc mesenteroides CMG713.Int. J. Biol. Sci. 2008; 4: 379-386Crossref Scopus (119) Google Scholar); nutrients in the form of amino acids, nucleotides, vitamins, and organic acids (Adrio and Demain, 2010Adrio J.L. Demain A.L. Recombinant organisms for production of industrial products.Bioeng. Bugs. 2010; 1: 116-131Crossref PubMed Scopus (104) Google Scholar); and pharmaceuticals themselves including insulin (Baeshen et al., 2014Baeshen N.A. Baeshen M.N. Sheikh A. Bora R.S. Ahmed M.M.M. Ramadan H.A.I. Saini K.S. Redwan E.M. Cell factories for insulin production.Microb. Cell Fact. 2014; 13: 141Crossref PubMed Scopus (164) Google Scholar), chemotherapeutics (Łukasiewicz and Fol, 2018Łukasiewicz K. Fol M. Microorganisms in the Treatment of Cancer: Advantages and Limitations.J. Immunol. Res. 2018; 2018: 2397808https://doi.org/10.1155/2018/2397808Crossref Scopus (35) Google Scholar) and antibiotics (Clardy et al., 2009Clardy J. Fischbach M.A. Currie C.R. The natural history of antibiotics.Curr. Biol. 2009; 19: R437-R441Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). In addition to their use as biological factories, the advent of synthetic biology has allowed microbes to be manipulated by humans to facilitate desirable processes, such as the bio-remediation of polluted environments and for use as living therapeutics. Microbial bio-remediation generally involves the breakdown of an undesirable substrate, rather than concentrating on the production of a functional product. Applications include sewage treatment (Dhall et al., 2012Dhall P. Kumar R. Kumar A. Biodegradation of sewage wastewater using autochthonous bacteria.ScientificWorldJournal. 2012; 2012: 861903Crossref PubMed Scopus (28) Google Scholar), removal of pesticides and aromatic compounds (McClure and Venables, 1986McClure N.C. Venables W.A. Adaptation of Pseudomonas putida mt-2 to growth on aromatic amines.J. Gen. Microbiol. 1986; 132: 2209-2218Google Scholar) from soil, and clean-up of oil spills (Mapelli et al., 2017Mapelli F. Scoma A. Michoud G. Aulenta F. Boon N. Borin S. Kalogerakis N. Daffonchio D. Biotechnologies for Marine Oil Spill Cleanup: Indissoluble Ties with Microorganisms.Trends Biotechnol. 2017; 35: 860-870Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Although microbes have been consciously used as therapeutics for over a century (Coley, 1991Coley W.B. The treatment of malignant tumors by repeated inoculations of erysipelas: With a report of ten original cases. 1893.Clin. Orthop. Relat. Res. 1991; : 3-11PubMed Google Scholar), the last decade has seen a plethora of possible applications presented. A non-exhaustive list includes diagnostic tools (Kotula et al., 2014Kotula J.W. Kerns S.J. Shaket L.A. Siraj L. Collins J.J. Way J.C. Silver P.A. Programmable bacteria detect and record an environmental signal in the mammalian gut.Proc. Natl. Acad. Sci. USA. 2014; 111: 4838-4843Crossref PubMed Scopus (226) Google Scholar, Riglar et al., 2017Riglar D.T. Giessen T.W. Baym M. Kerns S.J. Niederhuber M.J. Bronson R.T. Kotula J.W. Gerber G.K. Way J.C. Silver P.A. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation.Nat. Biotechnol. 2017; 35: 653-658Crossref PubMed Scopus (180) Google Scholar) and treatments for the human digestive system (Braat et al., 2006Braat H. Rottiers P. Hommes D.W. Huyghebaert N. Remaut E. Remon J.P. van Deventer S.J. Neirynck S. Peppelenbosch M.P. Steidler L. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease.Clin. Gastroenterol. Hepatol. 2006; 4: 754-759Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar), a treatment for oral mucositis (Caluwaerts et al., 2010Caluwaerts S. Vandenbroucke K. Steidler L. Neirynck S. Vanhoenacker P. Corveleyn S. Watkins B. Sonis S. Coulie B. Rottiers P. AG013, a mouth rinse formulation of Lactococcus lactis secreting human Trefoil Factor 1, provides a safe and efficacious therapeutic tool for treating oral mucositis.Oral Oncol. 2010; 46: 564-570Crossref PubMed Scopus (87) Google Scholar), HIV prevention (Lagenaur et al., 2011Lagenaur L.A. Sanders-Beer B.E. Brichacek B. Pal R. Liu X. Liu Y. Yu R. Venzon D. Lee P.P. Hamer D.H. Prevention of vaginal SHIV transmission in macaques by a live recombinant Lactobacillus.Mucosal Immunol. 2011; 4: 648-657Crossref PubMed Scopus (128) Google Scholar), and cancer immunotherapy (Zheng et al., 2017Zheng J.H. Nguyen V.H. Jiang S.N. Park S.H. Tan W. Hong S.H. Shin M.G. Chung I.J. Hong Y. Bom H.S. et al.Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin.Sci. Transl. Med. 2017; 9: eaak9537Crossref PubMed Scopus (252) Google Scholar). Various reviews have covered the novel applications that synthetic biology allows for bioremediation (de Lorenzo, 2009de Lorenzo V. Recombinant bacteria for environmental release: what went wrong and what we have learnt from it.Clin. Microbiol. Infect. 2009; 15: 63-65Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Pieper and Reineke, 2000Pieper D.H. Reineke W. Engineering bacteria for bioremediation.Curr. Opin. Biotechnol. 2000; 11: 262-270Crossref PubMed Scopus (333) Google Scholar, Sayler and Ripp, 2000Sayler G.S. Ripp S. Field applications of genetically engineered microorganisms for bioremediation processes.Curr. Opin. Biotechnol. 2000; 11: 286-289Crossref PubMed Scopus (222) Google Scholar) and for living therapeutics (Riglar and Silver, 2018Riglar D.T. Silver P.A. Engineering bacteria for diagnostic and therapeutic applications.Nat. Rev. Microbiol. 2018; 16: 214-225Crossref PubMed Scopus (165) Google Scholar). Despite the broad acceptance that there has been no known instance of a transgenic microbe being conferred a fitness advantage in the wild, frequent release of transgenic microbes into real world environments presents the possibility of such an individual manifesting. The responsibility of international governing bodies to understand and mitigate this potential problem has been established and ratified in legal treaties such as the Cartagena Protocol and the Rio Declaration on Environment and Development. Transgenic microbes capable of proliferating outside of a lab would lead to their uncontrolled propagation and persistence in an environment that may affect biological processes through interaction with indigenous populations, therefore reducing biodiversity and disrupting food webs. Increases in the risk of transfer of transgenic DNA through horizontal gene transfer could, in principle, lead to novel pathogens (NIH, 2019NIHNIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. https://osp.od.nih.gov/ufaq-category/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/.2019Google Scholar, Vermeire et al., 2015Vermeire T. Epstein M. Hartemann P. Proykova A. Rodriguez Farre E. Martinez Martinez L. Fernandes T. Chaudhry K. Chandra Rastogi S. Breitling R. et al.Opinion on Synthetic Biology II. Risk Assessment Methodologies and Safety Aspects. SCIENSANO.2015https://www.sciensano.be/en/biblio/opinion-synthetic-biology-ii-risk-assessment-methodologies-and-safety-aspectsGoogle Scholar). Although the full risks associated with the spread of transgenic microbes are not known, it is this very uncertainty that makes it important to implement control and regulation over transgenic microbial growth. Proving absolutely that a particular transgenic microbe capable of proliferation in the wild poses no environmental risk upon release is inherently an effort in futility, because biological processes contain too many unknown interactions for a perfectly predictive model to be formed. In cases where such uncertainty is prevalent, the precautionary principle can be applied to help form guidelines for the implementation of such a novel technology (Stirling, 2007Stirling A. Risk, precaution and science: towards a more constructive policy debate. Talking point on the precautionary principle.EMBO Rep. 2007; 8: 309-315Crossref PubMed Scopus (237) Google Scholar), with critics agreeing it can have a positive effect in such a situation (Peterson, 2007Peterson M. The precautionary principle should not be used as a basis for decision-making. Talking point on the precautionary principle.EMBO Rep. 2007; 8: 305-308Crossref Scopus (36) Google Scholar). For example, if a strain is developed capable of treating a Salmonella infection (Riglar et al., 2017Riglar D.T. Giessen T.W. Baym M. Kerns S.J. Niederhuber M.J. Bronson R.T. Kotula J.W. Gerber G.K. Way J.C. Silver P.A. Engineered bacteria can function in the mammalian gut long-term as live diagnostics of inflammation.Nat. Biotechnol. 2017; 35: 653-658Crossref PubMed Scopus (180) Google Scholar), it is prudent to introduce biological safeguards that would reduce the possibility of such a microbe escaping its intended environment (Stirling et al., 2017Stirling F. Bitzan L. O’Keefe S. Redfield E. Oliver J.W.K. Way J. Silver P.A. Rational Design of Evolutionarily Stable Microbial Kill Switches.Mol. Cell. 2017; 68: 686-697Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) even if we cannot fully predict what, if any, negative consequences may arise from such an escape. It is our view that transgenic microbes offer a range of solutions to environmental and health related issues, and in the coming decades they should and probably will be implemented. To facilitate this, exercising rigorous precaution will both increase public trust (Stirling et al., 2018Stirling A. Hayes K.R. Delborne J. Towards inclusive social appraisal: risk, participation and democracy in governance of synthetic biology.BMC Proc. 2018; 12: 15Crossref Scopus (20) Google Scholar) and, most importantly, reduce the possibility of negative consequences. For these reasons, the field of biocontainment has been of interest for decades. The Asilomar conference (Berg et al., 1975Berg P. Baltimore D. Brenner S. Roblin R.O. Singer M.F. Summary statement of the Asilomar conference on recombinant DNA molecules.Proc. Natl. Acad. Sci. USA. 1975; 72: 1981-1984Crossref PubMed Scopus (228) Google Scholar) laid out guidelines for the introduction of auxotrophies to prevent escape of transgenic microbes. With the advent of synthetic biology, it has been judged necessary to repeatedly revisit the topic, with a number of reviews appearing in the intervening years (Chari and Church, 2017Chari R. Church G.M. Beyond editing to writing large genomes.Nat. Rev. Genet. 2017; 18: 749-760Crossref PubMed Scopus (35) Google Scholar, Diwo and Budisa, 2018Diwo C. Budisa N. Alternative biochemistries for alien life: Basic concepts and requirements for the design of a Robust biocontainment system in genetic isolation.Genes (Basel). 2018; 10: E17Crossref PubMed Scopus (12) Google Scholar, Lee et al., 2018Lee J.W. Chan C.T.Y. Slomovic S. Collins J.J. Next-generation biocontainment systems for engineered organisms.Nat. Chem. Biol. 2018; 14: 530-537https://doi.org/10.1038/s41589-018-0056-xCrossref PubMed Scopus (104) Google Scholar, Moe-Behrens et al., 2013Moe-Behrens G.H.G. Davis R. Haynes K.A. Preparing synthetic biology for the world.Front. Microbiol. 2013; 4: 5Crossref PubMed Scopus (80) Google Scholar, Molin et al., 1993Molin S. Boe L. Jensen L.B. Kristensen C.S. Givskov M. Ramos J.L. Bej A.K. Suicidal genetic elements and their use in biological containment of bacteria.Annu. Rev. Microbiol. 1993; 47: 139-166Crossref PubMed Scopus (99) Google Scholar, Schmidt and de Lorenzo, 2012Schmidt M. de Lorenzo V. Synthetic constructs in/for the environment: managing the interplay between natural and engineered Biology.FEBS Lett. 2012; 586: 2199-2206Crossref PubMed Scopus (62) Google Scholar, Schmidt and De Lorenzo, 2016Schmidt M. De Lorenzo V. Synthetic bugs on the loose: Containment options for deeply engineered (micro)organisms.Curr. Opin. Biotechnol. 2016; 38: 90-96Crossref PubMed Scopus (46) Google Scholar, Torres et al., 2016Torres L. Krüger A. Csibra E. Gianni E. Pinheiro V.B. Synthetic biology approaches to biological containment: pre-emptively tackling potential risks.Essays Biochem. 2016; 60: 393-410Crossref PubMed Scopus (41) Google Scholar, Wright et al., 2013Wright O. Stan G.B. Ellis T. Building-in biosafety for synthetic biology.Microbiology. 2013; 159: 1221-1235Crossref PubMed Scopus (79) Google Scholar). The applications of transgenic microbes covered so far can broadly be divided into two categories; applications where the entire intended purpose can be carried out in a controlled setting and those where the intended purpose is designed to take place in an uncontrolled environment. Microbes engineered for production predominantly fall into the former category, whereas the majority of the second category are those microbes engineered to facilitate a process. However, both categories face the same requirements, that (1) transgenic material is not spread to other species, and (2) transgenic microbes are only viable in their intended environment. Containment systems to date predominantly address one or both of these two issues. A key means to quantify the effectiveness of a containment system used throughout this review is to measure the “survival ratio” it imparts on a population. The survival ratio is the term used to define the ratio of colony-forming units (CFU) in non-permissible conditions to the CFU in permissible conditions. For a survival ratio of 10−4, a population of 10,000 bacteria would yield only 1 survivor on transition to non-permissible conditions. Although not all systems can be quantified in such a manner—such as the essentializer in Stirling et al., 2017Stirling F. Bitzan L. O’Keefe S. Redfield E. Oliver J.W.K. Way J. Silver P.A. Rational Design of Evolutionarily Stable Microbial Kill Switches.Mol. Cell. 2017; 68: 686-697Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar—it is a useful tool for comparing different approaches and when assaying a systems effectiveness. In the United States, the National Institutes of Health has recommended a guideline providing that the “escape of the recombinant or synthetic nucleic acid molecule either via survival of the organisms or via transmission of the recombinant or synthetic nucleic acid molecule to other organisms should be less than 1 in 108 under specified conditions” (NIH, 2019NIHNIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. https://osp.od.nih.gov/ufaq-category/nih-guidelines-for-research-involving-recombinant-or-synthetic-nucleic-acid-molecules/.2019Google Scholar, Appendix 1-1-B). A survival ratio of 10−8 would currently meet these requirements. For many applications, it is likely that this threshold is too high, which is discussed further in “Containment standard practices.” Every transgenic organism constitutes an example of one or more units of genetic material existing in an organism or in an arrangement that does not exist in nature. This enables interactions and transfers of genetic material that have not previously been possible. The various strata of life display both unique and interconnected mechanisms for spreading genetic material, and the intentional transfer of such material by humans is not only possible, but performed on a routine basis. The potential negative consequences of this can often be categorized as low risk, like the expression of the GFP from the jellyfish Aequorea victoria. The use of GFP is considered harmless because no adverse consequences have been observed from its use in transgenic strains (Lee et al., 2018Lee J.W. Chan C.T.Y. Slomovic S. Collins J.J. Next-generation biocontainment systems for engineered organisms.Nat. Chem. Biol. 2018; 14: 530-537https://doi.org/10.1038/s41589-018-0056-xCrossref PubMed Scopus (104) Google Scholar, Moe-Behrens et al., 2013Moe-Behrens G.H.G. Davis R. Haynes K.A. Preparing synthetic biology for the world.Front. Microbiol. 2013; 4: 5Crossref PubMed Scopus (80) Google Scholar), and hypothetical problems that have been conceived of should transgenic strains expressing it be released are mild or uncertain. However, this by no means indicates that if GFP were spread throughout an environment it did not originate from, that it is impossible for it to have a negative impact. Because it would be essentially impossible to prove the innocuousness of the spread of a transgene in all conceivable scenarios, a more feasible and assured approach is to control its inability to spread in the first place with a reasonable level of confidence. Early attempts at preventing horizontal transfer of genetic material between microbes relied upon toxin-antitoxin systems expressed on plasmids and genomes (Díaz et al., 1994Díaz E. Munthali M. de Lorenzo V. Timmis K.N. Universal barrier to lateral spread of specific genes among microorganisms.Mol. Microbiol. 1994; 13: 855-861Crossref PubMed Scopus (62) Google Scholar, Torres et al., 2000Torres B. Jaenecke S. Timmis K.N. García J.L. Díaz E. A gene containment strategy based on a restriction-modification system.Environ. Microbiol. 2000; 2: 555-563Crossref PubMed Scopus (22) Google Scholar, Torres et al., 2003Torres B. Jaenecke S. Timmis K.N. García J.L. Díaz E. A dual lethal system to enhance containment of recombinant micro-organisms.Microbiology. 2003; 149: 3595-3601Crossref PubMed Scopus (47) Google Scholar) or host repression of a plasmid borne toxin (Knudsen and Karlström, 1991Knudsen S.M. Karlström O.H. Development of efficient suicide mechanisms for biological containment of bacteria.Appl. Environ. Microbiol. 1991; 57: 85-92Crossref PubMed Google Scholar) to prevent the spread of transgenic plasmids to other microbes. A similar approach has been proposed that uses origins of replication that are split between a plasmid and the host genome to prevent plasmid spreading (Wright et al., 2013Wright O. Stan G.B. Ellis T. Building-in biosafety for synthetic biology.Microbiology. 2013; 159: 1221-1235Crossref PubMed Scopus (79) Google Scholar). These two techniques were combined (Wright et al., 2015Wright O. Delmans M. Stan G.B. Ellis T. GeneGuard: A modular plasmid system designed for biosafety.ACS Synth. Biol. 2015; 4: 307-316Crossref PubMed Scopus (74) Google Scholar), achieving a survival ratio of <10−3 for each method individually, but the study did not report the survival ratio from the combined strain. These systems are summarized in Table 1. It is of note that the stability (see “Evolutionary stability”) of these systems (i.e., the capacity of the system to maintain its function when passaged in permissible conditions) is not reported for any system.Table 1Containment Systems Designed to Prevent the Spread of Transgenic PlasmidsYearLead AuthorSpeciesConditional OriginToxin/Antitoxin SystemSurvival RatioStability1991S.M. KnudsenE. coliRelF10−5not tested1994E. DiazE. colicolicin E310−4not tested2000B. TorresE. coliEcoRI10−4not tested2003B. TorresE. coliEcoRI, colicin E310−8not tested2014O. WrightE. coliColE2Kid or ζ<10−3not tested Open table in a new tab To achieve a “hard lock” (where the probability of the transfer and expression of transgenic material can be deemed low enough as to be negligible), it is necessary to address the “central dogma” of biology and unpick some of its core truths. Currently, all natural organisms are based around the same genetic code. The four deoxyribose nucleotides are transcribed into the four ribose nucleotides, which are in turn translated using the ubiquitous triplet codon code into the 20 standard amino acids that make up all proteins. Several studies have sought to alter this dynamic in one way or another. By engineering a tRNA synthetase, it has been shown that a quadruplet codon scheme can be implemented in order to incorporate non-natural amino acids (Chatterjee et al., 2014Chatterjee A. Lajoie M.J. Xiao H. Church G.M. Schultz P.G. A bacterial strain with a unique quadruplet codon specifying non-native amino acids.ChemBioChem. 2014; 15: 1782-1786Crossref PubMed Scopus (39) Google Scholar, Hankore et al., 2019Hankore E.D. Zhang L. Chen Y. Liu K. Niu W. Guo J. Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons.ACS Synth. Biol. 2019; 8: 1168-1174Crossref PubMed Scopus (15) Google Scholar). In addition, Escherichia coli has been engineered to survive on an artificial nucleotide (Marlière et al., 2011Marlière P. Patrouix J. Döring V. Herdewijn P. Tricot S. Cruveiller S. Bouzon M. Mutzel R. Chemical evolution of a bacterium’s genome.Angew. Chem. Int. Ed. Engl. 2011; 50: 7109-7114Crossref PubMed Scopus (147) Google Scholar) or to incorporate non-natural amino acid pairs (Malyshev et al., 2014Malyshev D.A. Dhami K. Lavergne T. Chen T. Dai N. Foster J.M. Corrêa Jr., I.R. Romesberg F.E. A semi-synthetic organism with an expanded genetic alphabet.Nature. 2014; 509: 385-388Crossref PubMed Scopus (378) Google Scholar). For containment applications, the most effective modification to the fundamental tenets of life upheld in the central dogma is to recode a genome so completely that it no longer requires the tRNA machinery for a specific codon or set of codons. This was first accomplished in bacteria with E. coli by removing all instances of the TAG stop codon and its corresponding tRNA (Lajoie et al., 2013Lajoie M.J. Rovner A.J. Goodman D.B. Aerni H.-R. Haimovich A.D. Kuznetsov G. Mercer J.A. Wang H.H. Carr P.A. Mosberg J.A. et al.Genomically recoded organisms expand biological functions.Science. 2013; 342: 357-360https://doi.org/10.1126/science.1241459Crossref PubMed Scopus (570) Google Scholar) and again, more recently, by removing TAG along with the serine encoding codons TCG and TCA (Fredens et al., 2019Fredens J. Wang K. de la Torre D. Funke L.F.H. Robertson W.E. Christova Y. Chia T. Schmied W.H. Dunkelmann D.L. Beránek V. et al.Total synthesis of Escherichia coli with a recoded genome.Nature. 2019; 569: 514-518Crossref PubMed Scopus (211) Google Scholar). Alternative methods have been published for the complete recoding of an organism such as an integrase-based approach to achieve a 57 codon E. coli genome (Ostrov et al., 2016Ostrov N. Landon M. Guell M. Kuznetsov G. Teramoto J. Cervantes N. Zhou M. Singh K. Napolitano M.G. Moosburner M. et al.Design, synthesis, and testing toward a 57-codon genome.Science. 2016; 353: 819-822Crossref PubMed Scopus (185) Google Scholar), a technique termed step-wise integration of rolling circle amplified segments (SIRCAS), used to remove two codons from the genome of Salmonella typhimurium (Lau et al., 2017Lau Y.H. Stirling F. Kuo J. Karrenbelt M.A.P. Chan Y.A. Riesselman A. Horton C.A. Schäfer E. Lips D. Weinstock M.T. et al.Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA.Nucleic Acids Res. 2017; 45: 6971-6980https://doi.org/10.1093/nar/gkx415Crossref PubMed Scopus (42) Google Scholar), and an extensive international effort in Saccharomyces cerevisiae that successfully removed all instances of the TAG stop codon (Mitchell et al., 2017Mitchell L.A. Wang A. Stracquadanio G. Kuang Z. Wang X. Yang K. Richardson S. Martin J.A. Zhao Y. Walker R. et al.Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond.Science. 2017; 355: eaaf4831Crossref Scopus (138) Google Scholar, Richardson et al., 2017Richardson S.M. Mitchell L.A. Stracquadanio G. Yang K. Dymond J.S. DiCarlo J.E. Lee D. Huang C.L. Chandrasegaran S. Cai Y. et al.Design of a synthetic yeast genome.Science. 2017; 355: 1040-1044Crossref PubMed Scopus (321) Google Scholar). The different recoding approaches are summarized in Table 2 and in previous reviews (Kuo et al., 2018Kuo J. Stirling F. Lau Y.H. Shulgina Y. Way J.C. Silver P.A. Synthetic genome recoding: new genetic codes for new features.Curr. Genet. 2018; 64: 327-333Crossref PubMed Scopus (14) Google Scholar).Table 2Recoding Approaches Attempted to DateYearLead AuthorSpecies% RecodedRecoding MethodCodons RemovedAmino Acid2011F. IsaacsE. coli100MAGE/CAGETAGStop2016N. OstrovE. coli100aThis was achieved over 87 different strains.integrase-based segments approachAGA, AGG, AGC, AGT, TTA, TTG, TAGArgSerLeuStop2017Y.H. LauS. typhimurium4.5SIRCASTTA, TTGLeu2017S. RichardsonbThe recoding of S. cerevisiae was a large collaborative effort that resulted in 7 publications in a special issue of Science (March 10, 2017), of which S. Richardson is one lead author.S. cerevisiae100SWaP-InTAGStop2019J. FredensE. coli100REXERTCG, TCA, TAGSerStopa This was achieved over 87 different strains.b The recoding of S. cerevisiae was a large collaborative effort that resulted in 7 publications in a special issue of Science (March 10, 2017), of which S. Richardson is one lead author. Open table in a new tab Freeing up a codon, and subsequently its associated tRNA, allows for four novel containment possibilities. First, a recoded organism could have transgenic genes that are only translated in a functional manner by the host by inserting a recoded stop codon throughout the open reading frame, thus initiating early termination of translation in any organism not recoded in the same manner (Figure 1A). It should be noted this approach cannot prevent the transfer of transgenic DNA and subsequent mutations that remove recoded codons would allow expression. Second, an approach could be to incorporate a broad range highly lethal toxin, such as an endonuclease, alongside any t" @default.
• W3026544066 created "2020-05-29" @default.
• W3026544066 creator A5071401517 @default.
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• W3026544066 date "2020-05-01" @default.
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• W3026544066 title "Controlling the Implementation of Transgenic Microbes: Are We Ready for What Synthetic Biology Has to Offer?" @default.
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• W3026544066 doi "https://doi.org/10.1016/j.molcel.2020.03.034" @default.
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