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Mol Cell. 2017 Nov 16;68(4):686-697.e3. doi: 10.1016/j.molcel.2017.10.033.

Rational Design of Evolutionarily Stable Microbial Kill Switches.

Author information

1
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.
2
Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Warren Alpert 536, Boston, MA 02115, USA.
3
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. Electronic address: pamela_silver@hms.harvard.edu.

Abstract

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented "essentializer" and "cryodeath" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after 10 days of growth in the mammalian gut.

KEYWORDS:

CspA; antitoxin; cold shock; containment; kill switch; lambda; library; promoter; synthetic biology; toxin

PMID:
29149596
PMCID:
PMC5812007
DOI:
10.1016/j.molcel.2017.10.033
[Indexed for MEDLINE]
Free PMC Article

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