Part:BBa_K3755046

Double Regulated(trp+lac) Kill Switch

An E.coli kill switch regulated by both lactose and tryptophan levels. High levels of lactose and tryptophan are needed to keep cells alive.

Introduction of hok/sok system

This kill switch is based on hok/sok toxin-antitoxin sytem. The hok/sok system is a type I toxin-antitoxin (TA) system of E.coli. In type I TA systems, the antitoxin is a small antisense RNA that inhibits the translation of small toxic proteins by binding to the corresponding mRNAs. The hok/sok system loci mainly consists of three parts, hok (host killing, Part:BBa_K3755043), sok (suppression of killing, Part:BBa_K3755045) and mok (modulation of killing). The hok gene encodes a toxic transmembrane protein, which can damage the membrane of E.coli. The reading frame of mok overlaps extensively with hok, and is necessary for expression and regulation of hok translation. The sok gene encodes a small cis-acting antisense RNA of 64 nucleotides which can attach with the mok mRNA leader region and inhibit its translation. Through inhibit the translation of mok, sok indirectly inhibits the expression of toxic proteins, hok.

Why we use hok/sok in our kill switch?

As most of the kill switches are based on type II toxin-antitoxin system, we choose hok/sok system. The antitoxin,sok is RNAs, but not proteins in our kill switch, which means that the producing of sok won't be a big burden for E.coli.

How does it work?

sok is expressed by T7 lac promoter. hok is expressed by trp promoter. When lactose(or IPTG) and tryptophan are both in the medium, the expression of sok will be strong while the expression of hok should be inhibited. So a great amount of sok will be able to inhibit the leaked expression of hok. When the E.coli escapes from the medium, expression of sok will reduce sharply, and the expression of hok will be activated. Then sok won't be able to inhibit the translation of hok. The E.coli will be killed by hok.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1406
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• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1406
  Illegal XbaI site found at 1903
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1406
  Illegal XbaI site found at 1903
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1670