Device
EXP-CBD_sy

Part:BBa_K5007008

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


Gal1-Kozak-CBD_syn-ADH1

Device designed as a transcriptional unit suitable for expression of the composite part CBD_syn, containing the coding sequences for the enzymes needed for the synthesis of cannabidiolic acid, the direct precursor to cannabidiol, an important pharmaceutical, from olivetolic acid and geranyl-diphosphate.

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
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 855
    Illegal BglII site found at 1317
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2961
    Illegal SapI site found at 2535

Design choices

This device consists of part BBa_K5007006, 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-k5007008-01.png
Figure 1: Diagram for part BBa_K5007008


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_K5007023 and BBa_K5007024 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

Luo X, Reiter MA, d'Espaux L, Wong J, Denby CM, Lechner A, Zhang Y, Grzybowski AT, Harth S, Lin W, Lee H, Yu C, Shin J, Deng K, Benites VT, Wang G, Baidoo EEK, Chen Y, Dev I, Petzold CJ, Keasling JD. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature. 2019 Mar;567(7746):123-126. doi: 10.1038/s41586-019-0978-9. Epub 2019 Feb 27. Erratum in: Nature. 2020 Apr;580(7802):E2. PMID: 30814733.

Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Shoyama Y, Morimoto S. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Lett. 2007 Jun 26;581(16):2929-34. doi: 10.1016/j.febslet.2007.05.043. Epub 2007 May 25. PMID: 17544411.

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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