Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain
Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain
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DOI：
10.1021/acssynbio.5b00085
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发表时间：
2015-12-01
影响因子：
4.7
通讯作者：
Anderson, J. Christopher
中科院分区：
文献类型：
--
作者：
Lopez, Gabriel;Anderson, J. Christopher
合成营养缺陷型生物是被工程化以需要特定分子的存在来维持生存力的生物。它们在生物控制和酶工程方面具有潜在的应用价值。我们表明，这些生物体可以通过将配体依赖性工程化到必需基因中来产生。我们展示了一种基于配体依赖性必需基因（SLiDE）的合成营养缺陷型的方法，使用5个必需基因作为测试用例：pheS，dnaN，tyrS，metG和adk。我们表明，当与配体苯并噻唑化学互补时，单个SLiDE菌株的活力可以增加1 × 10（8）倍。优化的SLiDE菌株工程方案需要不到1周和100美元。我们将多个SLiDE菌株等位基因组合到工业大肠杆菌菌株BL 21（DE 3）中，产生了超过生物安全标准的生物体，逃逸频率低于3 X 10（-11）的检测限。
Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. They have potential applications in biocontainment and enzyme engineering. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE) using 5 essential genes as test cases: pheS, dnaN, tyrS, metG, and adk. We show that a single SLiDE strain can have a 1 x 10(8)-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE strain engineering protocol required less than 1 week and $100 USD. We combined multiple SLiDE strain alleles into the industrial Escherichia coli strain BL21(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 X 10(-11).