The development of genetically engineered microbes (GEMs) has resulted in an urgent need to control their persistence in the environment. The use of biocontainment such as kill switches is a critical approach to prevent the unintended proliferation of GEMs; however, the effectiveness of kill switches─reported as escape rates, i.e., the ratio of the number of viable microbes when the kill switch is triggered relative to the number when it is not triggered─is typically assessed under laboratory conditions that do not resemble environmental conditions under which biocontainment must perform. In this study, we discovered that the escape rate of an Escherichia coli GEM biocontained with a CRISPR-based kill switch triggered by anhydrotetracycline (aTc) increased by 3–4 orders of magnitude when deployed in natural surface waters as compared to rich laboratory media. We identified that environmental conditions (e.g., pH, nutrient levels) may contribute to elevated escape rates in multiple ways, including by altering the chemical speciation of the kill switch trigger to reduce its uptake and providing limited nutrients required for the kill switch to function. Our study demonstrated that conditions in the intended environment must be considered in order to design effective GEM biocontainment strategies.

中文翻译：

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环境条件对生物封闭基因工程微生物逃逸率的影响


基因工程微生物 （GEM） 的发展导致迫切需要控制它们在环境中的持久性。使用生物防护装置（如终止开关）是防止 GEM 意外扩散的关键方法;然而，终止开关的有效性（报告为逃逸率，即触发终止开关时活菌数量与未触发时存活微生物数量之比）通常是在实验室条件下评估的，这些条件与必须执行生物防护的环境条件不同。在这项研究中，我们发现，与丰富的实验室培养基相比，当部署在天然地表水中时，由无水四环素 （aTc） 触发的基于 CRISPR 的杀伤开关生物包含的大肠杆菌 GEM 的逃逸率增加了 3-4 个数量级。我们发现环境条件 （例如，pH 值、营养水平） 可能以多种方式导致逃逸率升高，包括通过改变终止开关触发器的化学形态以减少其吸收，并提供终止开关发挥作用所需的有限营养。我们的研究表明，为了设计有效的 GEM 生物防护策略，必须考虑预期环境中的条件。