Part:BBa_K4321005

E.coli to Bacillus subtilis shuttle plasmid (pCG004)

pCG004 is an E.coli - Bacillus subtilis shuttle plasmid that was created based off of the pHT01 plasmid. This plasmid contains several features that can be used to selectively express desired genes inserted within this dropout region. The dropout region contains a green fluorescent protein (GFP) flanked by BsaI sites. Upstream of this region is the lacI sequence (encodes a lac repressor), and an overlapping lac operator (lacO) and pgrac promoter.

When IPTG is not present, the lac repressor (LacI) is expressed and bound to lacO. Due to the overlap between lacO and pGrac, downstream expression from Pgrac cannot occur due to promoter unavailability. This results in the repression of the downstream genes. When IPTG is present, it binds to LacI such that a conformational change occurs. This changes the shape of the lacO binding site in LacI and releases it from the lac operator. Since LacI is now removed, transcription factors now have access to Pgrac and the strong expression of the downstream genes can occur.

Usage and Biology

This plasmid can replicate in both E.coli and B.subtilis and can be used when B.subtilis is chosen as the destination bacteria. If insertion is in the dropout site, expression of the desired genes will be under the control of a strong IPTG responsive promoter, Pgrac.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 9048
  Illegal suffix found in sequence at 1
  Illegal EcoRI site found at 3615
  Illegal EcoRI site found at 6835
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3615
  Illegal EcoRI site found at 6835
  Illegal EcoRI site found at 9048
  Illegal NheI site found at 3451
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 9054
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3615
  Illegal EcoRI site found at 6835
  Illegal EcoRI site found at 9048
  Illegal BglII site found at 361
  Illegal BglII site found at 6306
  Illegal XhoI site found at 365
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 9048
  Illegal suffix found in sequence at 2
  Illegal EcoRI site found at 3615
  Illegal EcoRI site found at 6835
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 9048
  Illegal EcoRI site found at 3615
  Illegal EcoRI site found at 6835
  Illegal XbaI site found at 9063
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 6234
  Illegal BsaI.rc site found at 5170
  Illegal SapI.rc site found at 2338
  Illegal SapI.rc site found at 7459
  

Considerations

pCG004 contains a dropout green fluorescence protein (GFP) which is flanked by two BsaI sites. Replacement of this dropout GFP with our Cyt cassettes (K4321007 and K4321009) was achieved by BsaI digestion and ligation. Upstream of the dropout GFP is a strong IPTG inducible promoter, Pgrac. Insertion of desired genes into this dropout region will enable users to inducible control the expression of downstream genes.

Bacillus subtilis expression plasmids often have leaky expression in E.coli cells. This often results in the unexpected and oftentimes high expression of proteins. In this case, transformed DH5alpha E.coli cells fluorescent green due to the GFP mut3b absorption maxima of 501 nanometers, which falls in the white light spectrum.

References

PCG004 (plasmid #87377). Addgene. (n.d.). Retrieved October 8, 2022, from https://www.addgene.org/87377/

Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540