title: Intrinsic biocontainment: multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes.
abstract: Biocontainment may be required in a wide variety of situations such as work with pathogens, field release applications of engineered organisms, and protection of intellectual properties. Here, we describe the control of growth of the brewer's yeast, Saccharomyces cerevisiae, using both transcriptional and recombinational "safeguard" control of essential gene function. Practical biocontainment strategies dependent on the presence of small molecules require them to be active at very low concentrations, rendering them inexpensive and difficult to detect. Histone genes were controlled by an inducible promoter and controlled by 30 nM estradiol. The stability of the engineered genes was separately regulated by the expression of a site-specific recombinase. The combined frequency of generating viable derivatives when both systems were active was below detection (<10(-10)), consistent with their orthogonal nature and the individual escape frequencies of <10(-6). Evaluation of escaper mutants suggests strategies for reducing their emergence. Transcript profiling and growth test suggest high fitness of safeguarded strains, an important characteristic for wide acceptance.
authors.affiliation: School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom; School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; jef.boeke@nyumc.org yizhi.cai@ed.ac.uk.
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Cai
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Institute for Systems Genetics, NYU Langone Medical Center, New York, NY 10016; and.
authors.forename: Neta
authors.initials: N
authors.lastname: Agmon
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205;
authors.forename: Woo Jin
authors.initials: WJ
authors.lastname: Choi
authors.affiliation: School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom;
authors.forename: Alba
authors.initials: A
authors.lastname: Ubide
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Whiting School of Engineering, The Johns Hopkins University, Baltimore, MD 21218.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Stracquadanio
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205;
authors.forename: Katrina
authors.initials: K
authors.lastname: Caravelli
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205;
authors.forename: Haiping
authors.initials: H
authors.lastname: Hao
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Whiting School of Engineering, The Johns Hopkins University, Baltimore, MD 21218.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bader
authors.affiliation: School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Institute for Systems Genetics, NYU Langone Medical Center, New York, NY 10016; and jef.boeke@nyumc.org yizhi.cai@ed.ac.uk.
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D001709:Biotechnology / Q000379:methods*; D003264:Containment of Biohazards / Q000379:methods*; D020869:Gene Expression Profiling; D020043:Genes, Essential / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation / Q000235:genetics; D030781:Organisms, Genetically Modified / Q000235:genetics*; D045522:Recombinases / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D045522:Recombinases
keywords: Saccharomyces cerevisiae; biosafety; biotechnology