Plasmid
pCG004

Part:BBa_K4321005:Design

Designed by: Kulay Janneh   Group: iGEM22_Guelph   (2022-09-28)
Revision as of 21:02, 9 October 2022 by Kjanneh (Talk | contribs) (→‎Design Notes)


E.coli to Bacillus subtilis shuttle plasmid (pCG004)


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


Design Notes

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. 


      800px-PCG004_in_E_coli.jpeg

Source

pCG004 originates from the pHT01 plasmid, however this plasmid contains two alterations. The first is the removal of the backbone BsaI site within the AmpR cassette. Secondly, the multiple cloning site was replaced with a BsaI dropout consisting of a constitutive GFP mut3b cassette. This GFP cassette is expressed by the Pveg promoter, spoRBS and the rmB terminator. pCG004 was obtained directly from addgene.

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