Composite
CBD_sub

Part:BBa_K5007000:Design

Designed by: Pedro Ribeiro Faria   Group: iGEM23_USP-EEL-Brazil   (2023-09-23)


CsHCS1-T2A-CsOLS-T2A-CsOAC


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 228
    Illegal NgoMIV site found at 1531
    Illegal AgeI site found at 1090
    Illegal AgeI site found at 3271
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 771


Design Notes

The T2A self-cleaving peptide leaves fragments in the C-, N- or both protein terminals after translation and successful separation of each protein. The effect of these fragments on the final synthesized enzymes was evaluated through structural modeling, docking and several validation steps, so the parts' order in this composite is optimized in order for the T2A fragments to have the least interference with the individual proteins' predicted structure and function.

Additionally, this part can be assembled into different plasmids and devices, using either seamless cloning techniques, such as Gibson Assembly, or adequate restriction cloning, allowing for future iGEM teams to build up on this part.


Source

Part BBA_K1993019 came from iGEM16 SYSU-MEDICINE team, and this part comes from the porcine teschovirus-1. All other parts on this composite came from iGEM22 Pinene Express, and these parts come from the Cannabis Sativa plant.

References

Stout JM, Boubakir Z, Ambrose SJ, Purves RW, Page JE. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J. 2012 Aug;71(3):353-65. doi: 10.1111/j.1365-313X.2012.04949.x. Epub 2012 Jun 1. PMID: 22353623.

Taura F, Tanaka S, Taguchi C, Fukamizu T, Tanaka H, Shoyama Y, Morimoto S. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Lett. 2009 Jun 18;583(12):2061-6. doi: 10.1016/j.febslet.2009.05.024. Epub 2009 May 19. PMID: 19454282.

Gagne SJ, Stout JM, Liu E, Boubakir Z, Clark SM, Page JE. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proc Natl Acad Sci U S A. 2012 Jul 31;109(31):12811-6. doi: 10.1073/pnas.1200330109. Epub 2012 Jul 16. PMID: 22802619; PMCID: PMC3411943.

Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556.