Difference between revisions of "Part:BBa K1659201:Design"

 
 
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<partinfo>BBa_K1659201 short</partinfo>
 
<partinfo>BBa_K1659201 short</partinfo>
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===Design Notes===
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===Design Notes and Sources===
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to be updated
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We obtained the peptide sequence for DspB from Kaplan et al's original publication and converted it into a nucleotide sequence using [https://www.idtdna.com/CodonOpt IDT's Codon Optimizer], optimizing based on ''E. coli'' K-12's codon table [1].
  
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The gene sequence for DsbA 2-19 was obtained from [https://parts.igem.org/Part:BBa_K729004 BBa_K729004], a part comprising DsbA 2-19 fused to the N-terminal of micrococcal nuclease made by [http://2012.igem.org/Team:University_College_London Team UCL 2012]. The part sequence was not annotated, and as such we ran a [http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome Protein BLAST search] using the part's translated sequence to identify the bases that coded for our desired peptide. The sequence for DsbA 2-19 was adopted as-is, without further codon optimization.
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We fused the 2-19 peptide sequence of DsbA to the N-terminus of Dispersin B ([https://parts.igem.org/Part:BBa_K1659200 BBa_K1659200]) sequence without any spacer/linker peptide in between. A Hisx6 tag is added at the C-terminus for ease of protein purification using metal-affinity chromatography.
  
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===Source===
 
  
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to be updated
 
  
 
===References===
 
===References===
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[1] Kaplan, J.B. et al., 2004. Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms. Journal of Bacteriology, 186(24), pp.8213–8220.

Latest revision as of 08:53, 14 September 2015

Dispersin B fused at N-terminal with DsbA 2-19 signal sequence


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 316
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 472


Design Notes and Sources

We obtained the peptide sequence for DspB from Kaplan et al's original publication and converted it into a nucleotide sequence using IDT's Codon Optimizer, optimizing based on E. coli K-12's codon table [1].

The gene sequence for DsbA 2-19 was obtained from BBa_K729004, a part comprising DsbA 2-19 fused to the N-terminal of micrococcal nuclease made by [http://2012.igem.org/Team:University_College_London Team UCL 2012]. The part sequence was not annotated, and as such we ran a [http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome Protein BLAST search] using the part's translated sequence to identify the bases that coded for our desired peptide. The sequence for DsbA 2-19 was adopted as-is, without further codon optimization.

We fused the 2-19 peptide sequence of DsbA to the N-terminus of Dispersin B (BBa_K1659200) sequence without any spacer/linker peptide in between. A Hisx6 tag is added at the C-terminus for ease of protein purification using metal-affinity chromatography.


References

[1] Kaplan, J.B. et al., 2004. Genes Involved in the Synthesis and Degradation of Matrix Polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae Biofilms. Journal of Bacteriology, 186(24), pp.8213–8220.