Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, 3 Blackfan Circle, 5th Floor, Boston, MA 02115, USA.
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA.
Cell Rep. 2020 Mar 3;30(9):3139-3148.e4. doi: 10.1016/j.celrep.2020.02.033.
As pH is fundamental to all biological processes, pH-responsive bacterial genetic circuits enable precise sensing in any environment. Where the unintentional release of engineered bacteria poses a concern, coupling pH sensing to the expression of a toxin creates an effective bacterial containment system. Here, we present a pH-sensitive kill switch (acidic termination of replicating population [acidTRP]), based on the Escherichia coli asr promoter, with a survival ratio of <1 in 10. We integrate acidTRP with cryodeath to produce a 2-factor containment system with a combined survival ratio of <1 in 10 while maintaining evolutionary stability. We further develop a pulse-counting circuit with single-cell readout for each administered stimulus pulse. We use this pulse counter to record multiple pH changes and combine it with acidTRP to make a 2-count acid-sensitive kill switch. These results demonstrate the ability to build complex genetic systems for biological containment.
由于 pH 值对所有生物过程都至关重要，因此对 pH 值有响应的细菌遗传回路能够在任何环境中进行精确的感应。在工程细菌意外释放引起关注的情况下，将 pH 值感应与毒素的表达相结合，可创建有效的细菌遏制系统。在这里，我们提出了一种基于大肠杆菌 asr 启动子的 pH 敏感的杀伤开关（复制群体的酸性终止[acidTRP]），其存活率<1/10。我们将 acidTRP 与 cryodeath 集成在一起，形成了一种具有<1/10 的综合存活率的 2 因素遏制系统，同时保持进化稳定性。我们进一步开发了一种具有单细胞读出功能的脉冲计数电路，用于每个施用的刺激脉冲。我们使用此脉冲计数器记录多个 pH 值变化，并将其与 acidTRP 结合使用，制成 2 计数的 pH 敏感杀伤开关。这些结果表明了构建用于生物遏制的复杂遗传系统的能力。
