Composite

Part:BBa_K4999000:Design

Designed by: Piyush Nanda   Group: iGEM23_Harvard   (2023-10-11)


pBAD driven T4 lysis cassette


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
    Illegal NheI site found at 2560
    Illegal NheI site found at 2583
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2165
    Illegal AgeI site found at 2235
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961
    Illegal SapI site found at 2816


Design Notes

We had the following design consideration while designing this construct: 1. Adding anti-holin to the cassette to eliminate lysis due to leaky expression 2. Having araC in the same plasmid to avoid co-transforming two plasmids


Source

The T4 lysis cassette comprising of T4 endolysin, T4 holin and Anti-holin was synthesized from IDT as a gBlock. Ribosome binding site was added to the 5' region of cassette by PCR extension. The resulting construct was assembled into a pBAD plasmid that was received as a gift from Michael Laub lab (MIT).

References