Composite

Part:BBa_K5203008

Designed by: Guljahon Mustafokulova   Group: iGEM24_NYC-Empire-State   (2024-10-02)


perm-AgrC-AgrA-Sirius

This plasmid is used for 2024 NYC-Empire-State's AIP detection and response system. The AgrC codes for a sensor kinase on the cell membrane that responds to AIP signals in the environment when AIP binds to it on the membrane. This then causes the phosphorylation of AgrA, a response regulator in the signal transduction pathway, and AgrA will activate transcription. The Sirius is a blue fluorescent marker that signals AIP is being detected in the environment.

The Gibson assembly wizard was not able to pick primers for the AgrC-AgrA sequence and therefore the algorithm would not return any option. The different fragments and primers were therefore designed by hand, and the different parts were fed to the NEBuilder Tool for assembly of the final sequence.

The electrophoresis gel shows the failure of the Gibson assembly to form the new plasmids labeled. They are all the same size as the original pERM plasmid, which indicates the new fragments did not assemble properly into the plasmid.

This electrophoresis gel further indicates a failure as the bands are still the same size after correcting for proper ratios of DNA during the Gibson Assembly. Once again, there are no unique bands created when compared to the original pERM plasmid.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 401
    Illegal EcoRI site found at 4951
    Illegal XbaI site found at 4415
    Illegal PstI site found at 1153
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 401
    Illegal EcoRI site found at 4951
    Illegal NheI site found at 170
    Illegal PstI site found at 1153
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 401
    Illegal EcoRI site found at 4951
    Illegal BglII site found at 4393
    Illegal BamHI site found at 4958
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 401
    Illegal EcoRI site found at 4951
    Illegal XbaI site found at 4415
    Illegal PstI site found at 1153
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 401
    Illegal EcoRI site found at 4951
    Illegal XbaI site found at 4415
    Illegal PstI site found at 1153
    Illegal NgoMIV site found at 3479
    Illegal NgoMIV site found at 3833
    Illegal NgoMIV site found at 3993
    Illegal NgoMIV site found at 4361
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1327
    Illegal SapI.rc site found at 2409


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