Plasmid

Part:BBa_K5465999:Design

Designed by: Aritra Saha   Group: iGEM24_Oxford   (2024-10-02)


ONERING-TetO


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4843
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 4463
    Illegal NgoMIV site found at 4589
    Illegal NgoMIV site found at 4599
    Illegal AgeI site found at 4850
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4853
    Illegal BsaI.rc site found at 3280
    Illegal BsaI.rc site found at 3309
    Illegal BsaI.rc site found at 3652
    Illegal SapI site found at 3260
    Illegal SapI site found at 4491
    Illegal SapI site found at 5887


Design Notes

We chose pRPF144 backbone because it naturally contained high-copy number origins of replication for both gram positive and gram negative bacteria. This makes the TetR FROS system applicable to almost all bacteria types.

We also made sure that origins included were unlikely to be present in bacteria, so we can avoid any origin incompatibility

Our design consideration also resulted in including more than 10 TetO sequences in the TetO array to allow for sufficient localisations.

Additionally, using CatP as our antibiotic resistance selection marker, it prevented us from using bacteria which already have the resistance for the same antibiotic.


Source

pRPF144 backbone came from designated plasmid found here: https://www.addgene.org/106372/

Napari-Bacseg: https://github.com/piedrro/napari-bacseg Conj-Assay: https://github.com/alfbukys/conj_assay

References