Synthetic cassettes for pH-mediated sensing, counting and containment

Stirling, F.; Naydich, A.; Bramante, J.; Barocio, R.; Certo, M.; Wellington, H.; Redfield, E.; O'Keefe, S.; Gao, S.; Cusolito, A.; Way, J.; Silver, P. A.

doi:10.1101/740902

bioRxiv · 10.1101/740902

Synthetic cassettes for pH-mediated sensing, counting and containment

Stirling, F.·Naydich, A.·Bramante, J.·Barocio, R.·Certo, M.·Wellington, H.·Redfield, E.·O'Keefe, S.·Gao, S.·Cusolito, A.·Way, J.·Silver, P. A.

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Abstract

AbstractAs pH is fundamental to all biological processes, pH-responsive bacterial genetic circuits enable precise sensing in any environment. Where unintentional release of engineered bacteria poses a concern, coupling pH sensing to 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 E. coli asr promoter, with a survival ratio of less than 1 in 106. We integrate acidTRP with cryodeath to produce a two-factor containment system with a combined survival ratio of less than 1 in 1011 whilst 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 two-count acid-sensitive kill switch. These results demonstrate the ability to build complex genetic systems for biological containment.

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Stirling, F., Naydich, A., Bramante, J., Barocio, R., Certo, M., Wellington, H., Redfield, E., O'Keefe, S., Gao, S., Cusolito, A., Way, J., Silver, P. A.. 2019-08-20. Synthetic cassettes for pH-mediated sensing, counting and containment. https://doi.org/10.1101/740902

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