Plasmid

Part:BBa_K3697009

Designed by: Lauren Ramlan   Group: iGEM20_Stanford   (2020-10-23)
Revision as of 04:33, 25 October 2020 by Lramlan (Talk | contribs)


pVeg YFP Plasmid for B. subtilis

This part is an expression vector for Bacillus subtilis that produces codon-optimized YFP, for integration into Bacillus subtilis at the AmyE loci (BBa_K143001). YFP is under pVeg expression (BBa_K143012), the strongest constitutive promotor known in B. subtilis. Plasmid expresses kanamycin resistance in B. subtilis, and ampicillin resistance for cloning in E. coli.

YFP has an excitation peak is 514 nm and an emission peak is 527 nm.

This plasmid successfully integrates into B. subtilis and produces YFP at levels that can be quantified using a fluorescent plate reader. Transformation was performed using strains of xylose and mannitol inducible competent B. subtilis, and plated on selective kanamycin media (20ug/mL). When cloning in E. coli, the E. coli may be yellow, as they will non-discriminately express the YFP. E. coli transformed with this plasmid are a mild yellow under natural light for the duration of their lifetime and does not appear to degrade.

The RBS for expression of YFP in this vector is strong.

The YFP does not have a degradation tag.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4739
    Illegal XbaI site found at 1993
    Illegal SpeI site found at 1897
    Illegal PstI site found at 542
    Illegal PstI site found at 2138
    Illegal PstI site found at 4745
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4739
    Illegal SpeI site found at 1897
    Illegal PstI site found at 542
    Illegal PstI site found at 2138
    Illegal PstI site found at 4745
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4739
    Illegal BglII site found at 2010
    Illegal XhoI site found at 1643
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4739
    Illegal XbaI site found at 1993
    Illegal SpeI site found at 1897
    Illegal PstI site found at 542
    Illegal PstI site found at 2138
    Illegal PstI site found at 4745
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4739
    Illegal XbaI site found at 1993
    Illegal SpeI site found at 1897
    Illegal PstI site found at 542
    Illegal PstI site found at 2138
    Illegal PstI site found at 4745
    Illegal NgoMIV site found at 1679
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 716
    Illegal BsaI site found at 4001
    Illegal BsaI site found at 6371
    Illegal BsaI site found at 6403
    Illegal BsaI.rc site found at 6359
    Illegal BsaI.rc site found at 6391


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