Difference between revisions of "Part:BBa K4475006"

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===Enzyme and Product Detection===
 
===Enzyme and Product Detection===
  
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Assays were implemented for enzyme and product detection
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Assays were implemented for enzyme and product detection.
  
  

Revision as of 21:04, 13 October 2022


CTB3 Gene from Cercospora beticola

This is the full exon only (cDNA) coding sequence for the CTB3 enzyme in the fungus Cercospora beticola. The enzyme is the second in a multi step pathway for the production of cercosporin, a reactive oxygen species producing toxin in the presence of sunlight. FSU's iGEM team is looking to synthesize CTB3 in yeast as a proof of concept for cercosporin biosynthesis in a non-native and scalable organism, with the goal of producing cercosporin to combat algal blooms. The coding sequence has been codon optimized for Saccharomyces cerevisiae and modified to remove some illegal iGEM assembly cut sites via silent mutations. A 6x histidine tag is embedded on the N-terminus for expression verification.

Source: CB0940_00834 6-hydroxytryprostatin B O-methyltransferase [Cercospora beticola] https://www.ncbi.nlm.nih.gov/gene/35424646


Usage and Biology

CTB3 is a dual function O-methyltransferase and FAD- dependent monooxygenase [1] capable of converting nor-toralactone into cercoquinone C. The sequence was taken from genome of C. beticola and inserted into shuttle vectors for cloning in E.coli and episomal expression in S. cerevisiae.

Nor-ctb1.png

CTB1

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 3532
    Illegal BamHI site found at 3441
    Illegal BamHI site found at 5614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6121
    Illegal AgeI site found at 4195
  • 1000
    COMPATIBLE WITH RFC[1000]

Modulation of Design

In our project, choices of plasmid copy number and promoter strength were modulated to encourage maximum production of cercosporin intermediates in our chassis, resulting in several combinatorial designs. Molecular cloning was done using NEB HiFi DNA assembly, allowing DNA fragments for coding sequences and regulatory elements to be interchanged by annealing purposefully designed overlaps.

Enzyme and Product Detection

Assays were implemented for enzyme and product detection.


Enzyme Characterization

6xHis tagging was done for purification using liquid chromatography.


Product Characterization

Analytical chemistry assays for product identification and quantification were inspired by existing literature. (1,2,3)

References

(1) Newman AG, Townsend CA. Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae. J Am Chem Soc. 2016 Mar 30;138(12):4219-28. doi: 10.1021/jacs.6b00633. Epub 2016 Mar 16. PMID: 26938470; PMCID: PMC5129747.

(2) Adam G. Newman, Anna L. Vagstad, Philip A. Storm, and Craig A. Townsend. Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming. Journal of the American Chemical Society 2014 136 (20), 7348-7362 DOI: 10.1021/ja5007299

(3) de Jonge R, Ebert MK, Huitt-Roehl CR, Pal P, Suttle JC, Spanner RE, Neubauer JD, Jurick WM 2nd, Stott KA, Secor GA, Thomma BPHJ, Van de Peer Y, Townsend CA, Bolton MD. Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum. Proc Natl Acad Sci U S A. 2018 Jun 12;115(24):E5459-E5466. doi: 10.1073/pnas.1712798115. Epub 2018 May 29. Erratum in: Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):E8324. PMID: 29844193; PMCID: PMC6004482.