Difference between revisions of "Part:BBa M50437:Design"
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<partinfo>BBa_M50437 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_M50437 SequenceAndFeatures</partinfo>
  
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[[File:Plasmid map pLTK.png]]
  
 
===Design Notes===
 
===Design Notes===
  
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Our plasmid contained two distinct coding sequences, separately delineated with ribosome-binding sites.  
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This part contains an IPTG-inducible T5 promoter (Part:BBa_M50075) which was unmodified. By saturating cell media with IPTG, the T5 promoter can be fully induced in order to make an excess of ehpC and entC.
 +
This part also contains two strong ribosome binding sites: one before ahpC, and another before entC. The same strong RBS (part:BBa_M50080) was used twice, and it was used unmodified.
 +
We found the ahpC amino acid sequence on Uniprot and optimized the codon sequence for E. coli using the Integrated Data Technologies Codon Optimization Tool (IDT CodonOpt). We modified this sequence by adding FLAG (Part:BBa_T2004) onto the end of our ahpC sequence. This modified ahpC sequence is part BBa_M50435. Similarly, we acquired the entC amino acid sequence from UniProt and optimized the codon sequence for E. coli using IDT CodonOpt. We modified this by adding a 6xHis tag (Part:BBa_K112703) to the C-terminus. This modified entC sequence is part BBa_m50436 in the iGEM registry.
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Finally, we added the T7 Phage terminator downstream of our 6xHis tag (Part:BBa_M50060). This is a strong terminator for bacterial expression.
  
 
===Source===
 
===Source===
 +
Organism: All parts should be compatible with ''E. coli'' (some are endogenous)
  
−
T5 promoter: BBa_K784005
+
T5 promoter: BBa_M50075 <BR>
 +
Strong RBS: BBa_M50080 <BR>
 +
entC: BBa_M50436 <BR>
 +
ahpC: BBa_M50435 <BR>
 +
T7 terminator: BBa_M50080 <BR>
 +
 
 +
===References===
 +
 
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Frawley, E., & Fang, F., “The Ins and Outs of Bacterial Iron Metabolism.” Molecular Microbiology 2014. pp 609–616.Web. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135372/
 +
 
 +
Barghouthi, S., Payne,  S. M., Arcenaux, J. E. L., & Byers, B. R.  “Cloning, Mutagenesis, and Nucleotide Sequence of a Siderophore Biosynthetic Gene (amoA) from Aeromonas hydrophila.” Journal of Bacteriology, 1991. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC208203/pdf/jbacter00106-0221.pdf
 +
 
 +
Chamnoongpol, S., et al. “Fe(III)-mediated Cellular Toxicity.” Molecular Microbiology, 2002. Web https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.2002.03041.x
 +
 
 +
Ma, L. & Payne, S. “AhpC is Required for Optimal Production of Enterobactin by Escherichia coli”. Journal of Bacteriology, 2012. http://jb.asm.org/content/194/24/6748.long
 +
 
 +
Jacobs, A., White, G., Tait, G. “Iron Chelation in Cell Cultures by Two Conjugates of 2,3-DHB.” Biochemical and Biophysical Research Communications, 1977. Web. www.sciencedirect.com/science/article/pii/0006291X77906295
 +
 
 +
N. Ratledge, C., & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annual reviews in microbiology, 54(1), 881-941.

Latest revision as of 06:11, 12 June 2018

2,3-DHB Biosynthesis Construct, with entC and ahpC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 234
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 234
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 234
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 234
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 234
  • 1000
    COMPATIBLE WITH RFC[1000]

Plasmid map pLTK.png

Design Notes

This part contains an IPTG-inducible T5 promoter (Part:BBa_M50075) which was unmodified. By saturating cell media with IPTG, the T5 promoter can be fully induced in order to make an excess of ehpC and entC. This part also contains two strong ribosome binding sites: one before ahpC, and another before entC. The same strong RBS (part:BBa_M50080) was used twice, and it was used unmodified. We found the ahpC amino acid sequence on Uniprot and optimized the codon sequence for E. coli using the Integrated Data Technologies Codon Optimization Tool (IDT CodonOpt). We modified this sequence by adding FLAG (Part:BBa_T2004) onto the end of our ahpC sequence. This modified ahpC sequence is part BBa_M50435. Similarly, we acquired the entC amino acid sequence from UniProt and optimized the codon sequence for E. coli using IDT CodonOpt. We modified this by adding a 6xHis tag (Part:BBa_K112703) to the C-terminus. This modified entC sequence is part BBa_m50436 in the iGEM registry. Finally, we added the T7 Phage terminator downstream of our 6xHis tag (Part:BBa_M50060). This is a strong terminator for bacterial expression.

Source

Organism: All parts should be compatible with E. coli (some are endogenous)

T5 promoter: BBa_M50075
Strong RBS: BBa_M50080
entC: BBa_M50436
ahpC: BBa_M50435
T7 terminator: BBa_M50080

References

Frawley, E., & Fang, F., “The Ins and Outs of Bacterial Iron Metabolism.” Molecular Microbiology 2014. pp 609–616.Web. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135372/

Barghouthi, S., Payne, S. M., Arcenaux, J. E. L., & Byers, B. R. “Cloning, Mutagenesis, and Nucleotide Sequence of a Siderophore Biosynthetic Gene (amoA) from Aeromonas hydrophila.” Journal of Bacteriology, 1991. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC208203/pdf/jbacter00106-0221.pdf

Chamnoongpol, S., et al. “Fe(III)-mediated Cellular Toxicity.” Molecular Microbiology, 2002. Web https://onlinelibrary.wiley.com/doi/epdf/10.1046/j.1365-2958.2002.03041.x

Ma, L. & Payne, S. “AhpC is Required for Optimal Production of Enterobactin by Escherichia coli”. Journal of Bacteriology, 2012. http://jb.asm.org/content/194/24/6748.long

Jacobs, A., White, G., Tait, G. “Iron Chelation in Cell Cultures by Two Conjugates of 2,3-DHB.” Biochemical and Biophysical Research Communications, 1977. Web. www.sciencedirect.com/science/article/pii/0006291X77906295

N. Ratledge, C., & Dover, L. G. (2000). Iron metabolism in pathogenic bacteria. Annual reviews in microbiology, 54(1), 881-941.