Difference between revisions of "Part:BBa K4475000"
 
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CB0940_00833 CTB1 [Cercospora beticola]
 
CB0940_00833 CTB1 [Cercospora beticola]
 
https://www.ncbi.nlm.nih.gov/gene/35424645
 
https://www.ncbi.nlm.nih.gov/gene/35424645
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AlphaFold Structure - https://alphafold.ebi.ac.uk/entry/A0A2G5IC53
  
  
 
===Usage and Biology===
 
===Usage and Biology===
  
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This part is intended to be used as the first enzyme in the biosynthetic pathway of Cercosporin. It is intended to serve as an enzyme that can transform acetyl-CoA and 6 x malonyl-CoA into nor-toralactone.
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CTB1 is a multi-domain fungal polyketide synthase (2) capable of converting acetyl-CoA and 6 x malonyl-CoA into nor-toralactone.
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They were created from genome of C. beticola and sequentially inserted them into shuttle vectors for cloning in E.coli and episomal expression in S. cerevisiae.  
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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.  
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[[Image:CTB1.jpeg|400px|thumb|]]
  
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===CTB1===
  
 
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<partinfo>BBa_K4475000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4475000 SequenceAndFeatures</partinfo>
  
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===Modularity of Design===
  
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===Modulation of Design===
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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.
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+
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Choices of plasmid copy number and promoter strength were modulated to encourage maximum production 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.
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See: <br>
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BBa_K4475003 <br>
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BBa_K4475004 <br>
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BBa_K4475005 <br>
  
 
===Enzyme and Product Detection===
 
===Enzyme and Product Detection===
  
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Assays were then implemented for enzyme and product detection
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Assays were implemented for enzyme and product detection
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'''Characterization using HA Tagging'''
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'''Enzyme Characterization'''
  
 
HA epitope tagging was used.
 
HA epitope tagging was used.
  
  
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'''Characterization using HPLC'''
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'''Product Characterization'''
  
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This analytical chemistry assay was used for product identification and quantification inspired by existing literature. (1,2,3)
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The analytical chemistry assays proposed for product identification and quantification were inspired by existing literature. (1,2,3) <br>
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<P><b>Results will be added during the wiki melt after the jamboree.</b></P>
  
 
===References===
 
===References===

Latest revision as of 01:09, 14 October 2022


CTB1 Gene from Cercospora beticola

This is the full exon only (cDNA) coding sequence for the CTB1 enzyme in the fungus cercospora beticola. The enzyme is the first 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 CTB1 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. An HA tag is embedded on the N-terminus for expression verification.

Source: CB0940_00833 CTB1 [Cercospora beticola] https://www.ncbi.nlm.nih.gov/gene/35424645

AlphaFold Structure - https://alphafold.ebi.ac.uk/entry/A0A2G5IC53


Usage and Biology

CTB1 is a multi-domain fungal polyketide synthase (2) capable of converting acetyl-CoA and 6 x malonyl-CoA into nor-toralactone. 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.


CTB1.jpeg

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]

Modularity 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.

See:
BBa_K4475003
BBa_K4475004
BBa_K4475005

Enzyme and Product Detection

Assays were implemented for enzyme and product detection


Enzyme Characterization

HA epitope tagging was used.


Product Characterization

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

Results will be added during the wiki melt after the jamboree.

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.