Plasmid

Part:BBa_K3697010:Design

Designed by: Lauren Ramlan, Christopher Neimeth   Group: iGEM20_Stanford   (2020-10-22)


mCherry_BSU Plasmid


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4308
    Illegal XbaI site found at 1568
    Illegal SpeI site found at 1472
    Illegal PstI site found at 117
    Illegal PstI site found at 1713
    Illegal PstI site found at 3862
    Illegal PstI site found at 4314
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4308
    Illegal SpeI site found at 1472
    Illegal PstI site found at 117
    Illegal PstI site found at 1713
    Illegal PstI site found at 3862
    Illegal PstI site found at 4314
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4308
    Illegal BglII site found at 1585
    Illegal XhoI site found at 1218
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4308
    Illegal XbaI site found at 1568
    Illegal SpeI site found at 1472
    Illegal PstI site found at 117
    Illegal PstI site found at 1713
    Illegal PstI site found at 3862
    Illegal PstI site found at 4314
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4308
    Illegal XbaI site found at 1568
    Illegal SpeI site found at 1472
    Illegal PstI site found at 117
    Illegal PstI site found at 1713
    Illegal PstI site found at 3862
    Illegal PstI site found at 4314
    Illegal NgoMIV site found at 1254
    Illegal AgeI site found at 3532
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 291
    Illegal BsaI site found at 5940
    Illegal BsaI site found at 5972
    Illegal BsaI.rc site found at 5928
    Illegal BsaI.rc site found at 5960


Design Notes

This plasmid was already optimized for use in B. subtilis, but we had to consider which expression level and resistances were optimal for our purposes. We selected pVeg as the mCherry_BSU promotor due to its high expression levels in B. subtilis, allowing us to easily visualize the production of the mCherry reporter. We selected Kanamycin resistance in B. subtilis because KanR RNA was the target for the RNA toehold system we were testing. With KanR on the plasmid, the target for our detection system would be produced in the cell, and could be easily verified by plating on selective media.


Source

Pamela Silver Laboratory at Harvard Medical School

References