Composite

Part:BBa_K5181014:Design

Designed by: Mayukh Mitra and Eeshani Umesh Abhyankar   Group: iGEM24_IISER-TVM   (2024-09-03)


araBAD_RBS_SaSSY_FPPS


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 17
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1244
    Illegal BglII site found at 2615
    Illegal BglII site found at 4039
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1009
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2727
    Illegal SapI site found at 991
    Illegal SapI.rc site found at 1440
    Illegal SapI.rc site found at 3837


Design Notes

This is a bicistronic part designed such that gene SaSSy is positioned before FPPS for optimal expression, given that the P. putida genome codes for FPPS natively. After assembling 3 fragments, two cut sites, NcoI and NdeI, are formed.
The NcoI cut site (CCATGG) gets assembled in a way that makes the ATGG a part of the reading frame, starting at 6 bp after the first RBS. The NdeI cut site(CATATG) is a part of IRES, which gets formed after the assembly, and the 'ATG' marks the start of the reading frame 8 bp after the second RBS. Utility: Any gene starting with 'ATGG' can be placed under the arabinose promoter using the NcoI cut site in an experimentally verified ORF. One can use the IRES mentioned part BBa_K5181013 for a dual expression system and put their second gene in an experimentally verified ORF under the same promoter promoter using the NdeI cut site. The final part is assembled by Gibson assembly. The insert was PCR amplified using primers.

Forward primer:5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGG 3’

Reverse primer:5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA TGA CAA CTT GAC GGC TAC ATC ATT CAC 3’


pSEVA631, pSEVA241 and pSEVA424 with cut sites EcoRI and HindIII can be used as potential backbones for expression of this composite part.

Source

This part is a composite of all the following basic parts and can be used with pSEVA631 backbone: BBa_K5181004, BBa_K5181005, BBa_K5181006, BBa_K5181013.

References

1. The araBAD promoter sequence was obtained from Addgene pBbE8k-fcs.