Difference between revisions of "Part:BBa K2871000:Design"

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===Design Notes===
 
===Design Notes===
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The paper described by Charpentier & Oswald (2004), showed that a 20 amino acid signal sequence of Map protein was sufficient for translocation via E. coli T3SS. The short length signal sequence seemed attractive to us because we wanted to minimize folding interference between the signal sequence and the intended protein fused to the signal sequence.
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The amino acid sequence of the signal is obtained from Charpentier & Oswald (2004) paper. The DNA sequence is synthesized by IDT. The short glycine-serine linker (GGSGSGSG) is added by PCR to decrease folding interaction with a target protein. As this part was designed to be an N-terminal fusion domain, the RFC25 suffix sequence is added to the standard RFC10 suffix, which allows in-frame fusion with other RFC25 protein parts. The registry will recognize it as incompatible with RFC25 because of AgeI site on the RFC25 suffix that we added intentionally. But in fact, we can use it directly as an RFC25 part.  
  
 
===Source===
 
===Source===
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===References===
 
===References===
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Charpentier & Oswald, 2004. journal of baceteriology. 'Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter' DOI: 10.1128/JB.186.16.5486-5495.2004  
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Charpentier & Oswald, 2004. Journal of Bacteriology. 'Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter' DOI: 10.1128/JB.186.16.5486-5495.2004  
 
https://jb.asm.org/content/186/16/5486
 
https://jb.asm.org/content/186/16/5486

Latest revision as of 03:29, 18 October 2018


Map20, T3SS export signal peptide from Map gene


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 85
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The amino acid sequence of the signal is obtained from Charpentier & Oswald (2004) paper. The DNA sequence is synthesized by IDT. The short glycine-serine linker (GGSGSGSG) is added by PCR to decrease folding interaction with a target protein. As this part was designed to be an N-terminal fusion domain, the RFC25 suffix sequence is added to the standard RFC10 suffix, which allows in-frame fusion with other RFC25 protein parts. The registry will recognize it as incompatible with RFC25 because of AgeI site on the RFC25 suffix that we added intentionally. But in fact, we can use it directly as an RFC25 part.

Source

Synthetic sequence deduced from Amino acid Sequence from the Enteropathogenic E. coli strain E22. Described in the paper Charpentier & Oswald, 2004.

References

Charpentier & Oswald, 2004. Journal of Bacteriology. 'Identification of the Secretion and Translocation Domain of the Enteropathogenic and Enterohemorrhagic Escherichia coli Effector Cif, Using TEM-1 β-Lactamase as a New Fluorescence-Based Reporter' DOI: 10.1128/JB.186.16.5486-5495.2004 https://jb.asm.org/content/186/16/5486