Part:BBa_K305011

Subtilin inducible expression plasmid for Bacillus subtilis

Adapted from the system for subtilin-regulated gene expression (SURE) in Bacillus subtilis Bongers, this plasmid was constructed to express proteins present in RCF 10 BioBricks, composed of an RBS followed by a coding sequence, upon subtilin induction.

Usage and Biology

The SURE system is a stringently controlled expression system that displays both high levels of expression and very little promoter 'leakage'. This makes it ideal for the optimization of heterologous protein expression. The subtilin producing 'B. subtilis' ATCC 6633 strain senses the presense of the lantibiotic subtilin through the sensor histidine kinase SpaK, which phosphorylates its response regulator SpaR. SpaR can then bind to so-called spa boxes in the promoter regions of genes involved in subtilin biosynthesis, resulting in autoinduction of this system Kleerebezem. In the SURE system, a B. subtilis strain naturally lacking the subtilin biosynthesis and regulation genes has the spaRK genes introduced into its genome. A plasmid carrying a spa box promoter that is transformed to this strain can then drive the expression of proteins upon subtilin induction of SpaRK signalling.

Note that this expression backbone can only be used in a B. subtilis strain that has the spaRK genes integrated into its genome, i.e. B. subtilis NZ8900 used by Bongers et al, 2005 Bongers. Also, a subtilin producing strain of B. subtilis (B. subtilis ATCC 6633 used by Bongers et al, 2005) is required, of which the culture supernatant is used for induction of expression.

This backbone has issues when used in certain E. coli strains. See part design.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3141
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 3147
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 3141
  Illegal BglII site found at 3060
  Illegal XhoI site found at 287
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 3141
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 3141
  Plasmid lacks a suffix.
  Illegal XbaI site found at 3156
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SapI site found at 324
  

Part Characterization

Fluorescence of GFP part BBa_E0240 plotted against different concentrations of subtilin induction in liquid medium. These results demonstrate that addition of 0.5 to 1%(vol/vol) of subtilin to the culture is sufficient to reach optimal induction.

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References

<biblio>

1. Bongers pmid=16332878
2. Kleerebezem pmid=15374645

</biblio>