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Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes
Tuesday, 2015/02/17 | 06:09:42

Yizhi Caia,b,1, Neta Agmonb,c, Woo Jin Choib, Alba Ubidea, Giovanni Stracquadaniob,d, Katrina Caravellib, Haiping Haob, Joel S. Baderb,d, and Jef D. Boekeb,c,1


The advance of biotechnology opens up greater possibilities of bioterror and bioerror. Here, we propose multiplexed safeguard switches rooted in the development of foundational genomic, regulatory, and metabolic technologies. Safeguard switches can be regulated by submicromolar small molecule(s) and combined in a modular fashion. The resulting safeguard strains show high fitness and low reversion rates. Moreover, two distinct classes of safeguard switches are orthogonal, providing a potential fail-safe mechanism. The safeguard technologies provide a practical and generic approach to containing engineered microbes within defined laboratory and/or industrial environments, and can in principle be used in the field as well.


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.


See: http://www.pnas.org/content/112/6/1803.abstract.html?etoc

PNAS February 10, 2015 vol. 112 no. 6 1803-1808


Fig. 1.

Fig. 1. The structure of triplex histone switch. Duplex integrated safeguard based on a pair of histone genes. Histone H3 gene HHTS is regulated by a galactose promoter pGAL1, and the histone H4 gene HHFS is controlled by another galactose promoter pGAL7. Each histone switch is flanked by a pair of loxP sites. The duplex histone safeguard switches can either be on a CEN/ARS plasmid (A) or integrated into a genomic locus (B). Either the native Gal4 protein or an engineered tribrid protein called GEV turns on the transcription of the histone genes in the presence of their ligands, such that the engineered yeast survives. The third level of redundancy is use of an orthogonal control mechanism, site-specific recombination. Any pair of loxP sites (yellow diamonds) on the construct will recombine and delete one or both histone genes upon Cre, activation, leading to inviability. Both proteins can be independently controlled by small molecule(s). Hooked arrows, promoters; filled arrows, histone genes; blue lozenges, standardized vector components; HOL, HOR, sequences to Left and Right of HO gene, where the safeguard was integrated.

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