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Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment

2020; Cell Press; Volume: 30; Issue: 9 Linguagem: Inglês

10.1016/j.celrep.2020.02.033

2639-1856

Finn Stirling, Alexander D. Naydich, Juliet Bramante, Rachel Barocio, Michael Certo, Hannah Wellington, Elizabeth Redfield, Samuel O’Keefe, Sherry Shiqian Gao, Adam Cusolito, Jeffrey C. Way, Pamela A. Silver,

Bacterial Genetics and Biotechnology

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 106. We integrate acidTRP with cryodeath to produce a 2-factor containment system with a combined survival ratio of <1 in 1011 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.