Part:BBa_K4998044

Kill-switch device for B. subtilis WB800N

Usage and Biology

This device is a kill-switch cirquit for B. subtilis WB800N strain, that is regulated by the presence of IPTG in the medium. It consists of a lacI composite part (BBa_K1088019), the composite tetR part, BBa_K4998041 and the bsrG toxin transcriptional unit (BBa_K4998043).

Kill switch function

Alive state

This state is characterized by the presence of IPTG in the medium that surrounds the encapsulated Euphoresis microorganisms. IPTG binds to the LacI and prohibits it from suppressing TetR expression, whose gene expression is regulated by the Pgrac promoter, which contains LacO operator sites. This way, TetR is freely expressed and binds to the operator sites of the PlTetO promoter, which regulates the production of the toxin gene, bsrG, suppressing gene expression. In this manner, with the presence of IPTG in the medium, which means that Bacillus subtilis is still encapsulated in the microspheres inside the hydrogel, B. subtilis stays alive.

Deceased state

The IPTG reserves in the medium will be depleted before the hydrogel’s and the microspheres’ degradation. In the absence of IPTG, lacI will be able to bind to the lacO operator of the Pgrac promoter that regulates tetR production and suppress the expression of the tetR protein. This will lead to the production of the BsrG toxin, that will lead the B. subtilis cells into cell lysis, as there will be no tetR protein to bind to the operator sites of the PlTetO promoter, located before the toxin gene

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 2944
  Illegal suffix found in sequence at 12
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2944
  Illegal NheI site found at 2991
  Illegal NheI site found at 3014
  Illegal SpeI site found at 13
  Illegal PstI site found at 27
  Illegal NotI site found at 20
  Illegal NotI site found at 2950
  Illegal NotI site found at 4202
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2944
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 2944
  Illegal suffix found in sequence at 13
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 2944
  Illegal XbaI site found at 2959
  Illegal SpeI site found at 13
  Illegal PstI site found at 27
  Illegal NgoMIV site found at 1386
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 2184