Cai Yizhi, Agmon Neta, Choi Woo Jin, Ubide Alba, Stracquadanio Giovanni, Caravelli Katrina, Hao Haiping, Bader Joel S, Boeke Jef D
School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, United Kingdom; School of Medicine, The Johns Hopkins University, Baltimore, MD 21205;
School of Medicine, The Johns Hopkins University, Baltimore, MD 21205; Institute for Systems Genetics, NYU Langone Medical Center, New York, NY 10016; and.
Proc Natl Acad Sci U S A. 2015 Feb 10;112(6):1803-8. doi: 10.1073/pnas.1424704112. Epub 2015 Jan 26.
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.
在多种情况下可能都需要生物遏制，比如处理病原体、工程生物的野外释放应用以及知识产权保护等。在此，我们描述了利用对必需基因功能的转录和重组“保障”控制来控制酿酒酵母（Saccharomyces cerevisiae）的生长。依赖小分子存在的实用生物遏制策略要求它们在极低浓度下具有活性，这使得它们成本低廉且难以检测。组蛋白基因由一个可诱导启动子控制，并受30 nM雌二醇调控。工程基因的稳定性则通过位点特异性重组酶的表达单独调节。当两个系统都活跃时，产生有活力衍生物的综合频率低于检测限（<10^(-10)），这与其正交性质以及各自<10^(-6)的逃逸频率一致。对逃逸突变体的评估提出了减少其出现的策略。转录谱分析和生长测试表明受保障菌株具有高适应性，这是被广泛接受的一个重要特征。
