Composite

Part:BBa_K3848004:Design

Designed by: Atharv Salvi   Group: iGEM21_IISER_Bhopal   (2021-09-24)
Revision as of 13:24, 24 September 2021 by Kaizen (Talk | contribs) (→‎References)


Lactate regulated kill switch


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 78
    Illegal NheI site found at 101
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2457
    Illegal AgeI site found at 2527
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 391


Design Notes

The full sequence for the kill switch was codon optimized for E. coli. And the distance between the RBS and the start codons of the coding components of the part was adjusted to optimal length by adding GC-rich junk (non-coding) DNA. Also, the RBS for the antiholin and cI repressor is a medium strength RBS as these proteins delay the triggering of the kill switch and we do not need high amounts of these, as this will allow the bacteria to linger on for longer in non-tumor environments. Whereas, the RBS for the holin and endolysin expression is a strong RBS so as to express these proteins in a rapid manner to kill the cell in a short amount of time.


Source

Antiholin, holin and endolysin used in this part are derived from T4 bacteriophage. And the coding region of the cI repressor and the cI regulated promoter are derived from Lambda bacteriophage. All the other components are from E. coli.

References

1] de la Cruz-López, Karen G et al. “Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches.” Frontiers in oncology vol. 9 1143. 1 Nov. 2019, doi:10.3389/fonc.2019.01143

[2] Savva, Christos G et al. “The holin of bacteriophage lambda forms rings with large diameter.” Molecular microbiology vol. 69,4 (2008): 784-793. doi:10.1111/j.1365-2958.2008.06298.x