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Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection
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Rational Design of Evolutionarily Stable Microbial Kill Switches.

Finn Stirling1, Lisa Bitzan2, Samuel O'Keefe2

  • 1Department 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.

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Engineered "essentializer" and "cryodeath" synthetic genetic circuits function as stable bacterial kill switches. These circuits, designed for evolutionary stability, offer precise control over cell life and death in Escherichia coli.

Keywords:
CspAantitoxincold shockcontainmentkill switchlambdalibrarypromotersynthetic biologytoxin

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Area of Science:

  • Synthetic biology
  • Microbial genetics
  • Evolutionary engineering

Background:

  • Synthetic genetic circuits are crucial for understanding and applying genetic control.
  • Environmental control of life-and-death switches in cells presents a significant challenge.
  • Evolutionary stability is paramount for the reliable function of genetic elements.

Purpose of the Study:

  • To design and evaluate evolutionarily stable synthetic genetic kill switches.
  • To develop novel genetic circuits for controlling cell viability in Escherichia coli.
  • To assess the long-term functionality and stability of engineered genetic systems.

Main Methods:

  • Rational design principles were applied to engineer genetic circuits.
  • A toxin/antitoxin titering approach was used for construct screening.
  • The functional stability of kill switches was tested in vitro and in vivo.

Main Results:

  • Two kill switch circuits, 'essentializer' and 'cryodeath', were successfully developed.
  • Both circuits demonstrated sustained functionality for over 140 generations in vitro.
  • The cryodeath circuit effectively controlled population growth in the mammalian gut with low escape frequency (<1 in 10^5).

Conclusions:

  • Engineered essentializer and cryodeath circuits represent evolutionarily stable genetic kill switches.
  • These synthetic circuits offer robust control over cell viability in microbial populations.
  • The cryodeath system shows promise for applications requiring precise environmental control in vivo.