Difference between revisions of "Part:BBa K3407017:Design"
 
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===Design Notes===
 
===Design Notes===
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The Cry protein, Cry7Ca1 (GenBank accession no. EF486523) from the Bt strain BTH-13 is a three-domain Cry protein with a molecular weight of 129,196.50 kDa, whereas the activated toxin has a molecular weight of 68 kDa (Figure 2). The Cry7Ca1 toxin (activated Cry7Ca1 protein) crystal structure has been published and can be found in the Protein Data Bank under the accession number 5ZI1 <html><a href="#1">[1]</a></html>.  
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The Cry protein, Cry7Ca1 (GenBank accession no. EF486523) from the Bt strain BTH-13 is a three-domain Cry protein with a molecular weight of 129,196.50 kDa, whereas the activated toxin has a molecular weight of 68 kDa. This part contains the activated toxin. The gene was codon-optimized for expression in <i>Escherichia coli</i> using the GenSmartTM Codon Optimization Tool. The forbidden restriction enzymes sites are the following: BioBrick forbidden restriction sites (EcoRI, XbaI, SpeI, PstI) and Type IIS forbidden restriction sites (BsaI, SapI and Bpil).
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The Cry7Ca1 toxin gene was codon-optimized for Escherichia coli using the GenSmartTM Codon Optimization Tool (https://www.genscript.com/tools/gensmart-codon-optimization). The forbidden restriction enzymes sites are the following:
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- BioBrick forbidden restriction sites: EcoRI, XbaI, SpeI, PstI
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- Type IIS forbidden restriction sites: BsaI, SapI and Bpil
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The expression cassette of this part was synthesized in the backbone vector pTWIST_Amp_HighCopy from Twist bioscience, containing the viral promoter and terminator from the T7 bacteriophage. This synthetically synthetised plasmid is referred as pTWIST_Cry7Ca1.
  
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This part was designed as if it was cloned using Modular Cloning Toolkit <html><a href="#1">[1]</a></html><html><a href="#2">[2]</a></html>, so it contians the scars left after assembly.
  
 
===Source===
 
===Source===

Latest revision as of 23:12, 27 October 2020


Cry7Ca1 toxin with inducible T7 promoter, RBS and T7 terminator


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 688
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1388
    Illegal XhoI site found at 367
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Design Notes

The Cry protein, Cry7Ca1 (GenBank accession no. EF486523) from the Bt strain BTH-13 is a three-domain Cry protein with a molecular weight of 129,196.50 kDa, whereas the activated toxin has a molecular weight of 68 kDa. This part contains the activated toxin. The gene was codon-optimized for expression in Escherichia coli using the GenSmartTM Codon Optimization Tool. The forbidden restriction enzymes sites are the following: BioBrick forbidden restriction sites (EcoRI, XbaI, SpeI, PstI) and Type IIS forbidden restriction sites (BsaI, SapI and Bpil).

The expression cassette of this part was synthesized in the backbone vector pTWIST_Amp_HighCopy from Twist bioscience, containing the viral promoter and terminator from the T7 bacteriophage. This synthetically synthetised plasmid is referred as pTWIST_Cry7Ca1.

This part was designed as if it was cloned using Modular Cloning Toolkit [1][2], so it contians the scars left after assembly.

Source

Synthetically synthesised.


References

Ordered List

  1. Jing, X., Yuan, Y., Wu, Y., Wu, D., Gong, P., & Gao, M. (2019). Crystal structure of Bacillus thuringiensis Cry7Ca1 toxin active against Locusta migratoria manilensis. Protein science : a publication of the Protein Society, 28(3), 609–619.