Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment
Finn Stirling,
Alexander Naydich,
Juliet Bramante,
Rachel Barocio,
Michael Certo,
Hannah Wellington,
Elizabeth Redfield,
Samuel O’Keefe,
Sherry Gao,
Adam Cusolito,
Jeffrey Way,
Pamela Silver
Affiliations
Finn Stirling
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
Alexander Naydich
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
Juliet Bramante
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Rachel Barocio
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Michael Certo
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
Hannah Wellington
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Elizabeth Redfield
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Samuel O’Keefe
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Sherry Gao
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Adam Cusolito
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA
Jeffrey Way
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
Pamela Silver
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; Corresponding author
Summary: 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. : Stirling et al. develop a containment system for bacteria that responds to temperature and pH, allowing <1 in 1011 members of a population to escape. In addition, they develop a system that counts exposures to an environmental signal that will only produce an output if 2 distinct signals are registered. Keywords: containment, library, synthetic biology, lambda, CspA, asr, promoter, toxin, antitoxin, pH, counting, kill switch, pulse, falling edge
