title: Multilayered genetic safeguards limit growth of microorganisms to defined environments.
abstract: Genetically modified organisms (GMOs) are commonly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental settings require enhanced safety and security measures. Intrinsic biocontainment, the creation of bacterial hosts unable to survive in natural environments, remains a major unsolved biosafety problem. We developed a new biocontainment strategy containing overlapping 'safeguards'-engineered riboregulators that tightly control expression of essential genes, and an engineered addiction module based on nucleases that cleaves the host genome-to restrict viability of Escherichia coli cells to media containing exogenously supplied synthetic small molecules. These multilayered safeguards maintain robust growth in permissive conditions, eliminate persistence and limit escape frequencies to <1.3 × 10(-12). The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and undomesticated species.
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
authors.forename: Ryan R
authors.initials: RR
authors.lastname: Gallagher
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
authors.forename: Jaymin R
authors.initials: JR
authors.lastname: Patel
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA.
authors.forename: Alexander L
authors.initials: AL
authors.lastname: Interiano
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA.
authors.forename: Alexis J
authors.initials: AJ
authors.lastname: Rovner
authors.affiliation: Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06520, USA Systems Biology Institute, Yale University, West Haven, CT 06516, USA farren.isaacs@yale.edu.
authors.forename: Farren J
authors.initials: FJ
authors.lastname: Isaacs
mesh_terms: D003001:Cloning, Molecular; D018920:Coculture Techniques; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D030781:Organisms, Genetically Modified / Q000254:growth & development*; D011995:Recombination, Genetic
chemical_list: D003470:Culture Media