TY - JOUR
T1 - Intrinsic biocontainment
T2 - Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes
AU - Cai, Yizhi
AU - Agmon, Neta
AU - Choi, Woo Jin
AU - Ubide, Alba
AU - Stracquadanio, Giovanni
AU - Caravelli, Katrina
AU - Hao, Haiping
AU - Bader, Joel S.
AU - Boeke, Jef D.
PY - 2015/2/10
Y1 - 2015/2/10
N2 - 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 derivativeswhen both systemswere 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.
AB - 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 derivativeswhen both systemswere 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.
KW - Biosafety
KW - Biotechnology
KW - Saccharomyces cerevisiae
UR - http://www.scopus.com/inward/record.url?scp=84922678329&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84922678329&partnerID=8YFLogxK
U2 - 10.1073/pnas.1424704112
DO - 10.1073/pnas.1424704112
M3 - Article
C2 - 25624482
AN - SCOPUS:84922678329
SN - 0027-8424
VL - 112
SP - 1803
EP - 1808
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 6
ER -