Composite

Part:BBa_K5201002:Design

Designed by: YAU LI, Shun Lam Wong   Group: iGEM24_HongKong-UCCKE   (2024-09-30)


pLac-B0010-pmHAS*1-703-kfiD-B0015


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 957
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2592
    Illegal BamHI site found at 3124
    Illegal XhoI site found at 3467
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1456


Design Notes

Lac promoter (BBA_R0010) is chosen since it is an inducible promoter which can be induced by lactose or isopropyl β-D-1-thiogalactopyranoside (IPTG).

To further verify desirable protein is produced from the plasmid, affinity chromatography can be performed. 6X-his tag was placed in front of the coding sequence, to allow proteins to be purified and be detected.

We express both pmHAS and kfiD under the same Lac promoter by including an internal RBS. This can prevent promoter competition and express both proteins in a similar ratio.



Source

pmHAS : Pasturella Multocida kfiD : E. coli K5 strain

References