Device
EXP-CBD_su

Part:BBa_K5007007

Designed by: Pedro Ribeiro Faria   Group: iGEM23_USP-EEL-Brazil   (2023-10-03)


Gal1-Kozak-CBD_sub-ADH1

Device designed as a transcriptional unit suitable for expression of the composite part CBD_sub, containing the coding sequences for the enzymes needed for the intracellular synthesis of olivetolic acid, a key cannabinoid synthesis precursor, from hexanoic acid.

This device is designed for yeast chassis, and optimized for Saccharomyces cerevisiae. Assembled with no scars, suited for cloning and assembling into plasmids and other parts through Gibson Assembly.


Usage and Biology

Sequence and Features


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 777
    Illegal NgoMIV site found at 2080
    Illegal AgeI site found at 150
    Illegal AgeI site found at 1639
    Illegal AgeI site found at 3820
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1320

Design choices

This device consists of part BBa_K5007000, flanked on 5’ by BBa_J63006 and on 3’ by BBa_K392003. These parts form a transcriptional unit suitable for S. cerevisiae, in a composite capable of synthesizing three different enzymes from a single monocistronic mRNA.

bba-k5007007-01.png
Figure 1: Diagram for part BBa_K5007007


The Gal1 promoter being used was developed by the Silver Lab, and has shown efficacy in the transcription of fragments of different lengths and properties. The ADH1 terminator being used was tested by iGEM10 Osaka team in the transcript termination of three different constructs.

Part BBa_K5007000 can be obtained from this device by using the primer set BBa_K5007021 and BBa_K5007022 in PCR reactions, then cloned into different devices or biological circuits. This interchangeability of the entire coding sequence was designed so multiple teams can experiment with this part.

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.

Flick, J. S., & Johnston, M. (1990). Two Systems of Glucose Repression of the GAL] Promoter in Saccharomyces cerevisiae. MOLECULAR AND CELLULAR BIOLOGY, 10(9), 4757–4769.

Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/SB500366V

Curran KA, Karim AS, Gupta A, Alper HS. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metab Eng. 2013 Sep;19:88-97. doi: 10.1016/j.ymben.2013.07.001. Epub 2013 Jul 12. PMID: 23856240; PMCID: PMC3769427.</p>


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