Difference between revisions of "Part:BBa K1351010:Design"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1351010 short</partinfo>
 
<partinfo>BBa_K1351010 short</partinfo>
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===Design Notes===
 
===Design Notes===
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none
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The cell wall-binding region was chosen according to Liew ''et. al.'', 2012.
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===Source===
 
===Source===
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This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3.  
 
This part was generated by amplification from ''B. subtilis'' W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3.  
  
−
CWAD_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCTGAAGCCAGTGATATTGAAGACCC  
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CWAD_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCTGAAGCCAGTGATATTGAAGACCC <br>
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+
 
CWAD_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TCACGTTCTGGAGGCGCTC
 
CWAD_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TCACGTTCTGGAGGCGCTC
 +
 +
A AgeI site was removed by changing A to G at position 150 without altering the protein sequence by Quikchange PCR with the following mutagenesis primers:<br>
 +
CWAD_Age150mut_fwd: GAAACTCCGGGAACAGCgCCGGTTCATCAGCTTCC <br>
 +
CWAD_Age150mut_rev: GGAAGCTGATGAACCGGcGCTGTTCCCGGAGTTTC
  
 
===References===
 
===References===
 +
Liew, P. X., C. L. Wang and S. L. Wong. "Functional Characterization and Localization of a Bacillus Subtilis Sortase and Its Substrate and Use of This Sortase System to Covalently Anchor a Heterologous Protein to the B. Subtilis Cell Wall for Surface Display." J Bacteriol 194, no. 1 (2012): 161-75. [http://www.ncbi.nlm.nih.gov/pubmed/22020651 PubMed]
 +
 +
Oussenko, I. A., R. Sanchez and D. H. Bechhofer. "Bacillus Subtilis Yhcr, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure." J Bacteriol 186, no. 16 (2004): 5376-83. [http://www.ncbi.nlm.nih.gov/pubmed/15292138 PubMed]

Latest revision as of 14:15, 5 October 2014

Cell wall-anchoring domain of B. subtilis endonuclease YhcR


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 81
    Illegal BamHI site found at 96
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 52


Design Notes

The cell wall-binding region was chosen according to Liew et. al., 2012.

Source

This part was generated by amplification from B. subtilis W168 gDNA with the primers listed below, followed by digestion with EcoRI and PstI and ligation into pSB1C3.

CWAD_YhcR_ ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGCATG GCTGAAGCCAGTGATATTGAAGACCC
CWAD_YhcR_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT TCACGTTCTGGAGGCGCTC

A AgeI site was removed by changing A to G at position 150 without altering the protein sequence by Quikchange PCR with the following mutagenesis primers:
CWAD_Age150mut_fwd: GAAACTCCGGGAACAGCgCCGGTTCATCAGCTTCC
CWAD_Age150mut_rev: GGAAGCTGATGAACCGGcGCTGTTCCCGGAGTTTC

References

Liew, P. X., C. L. Wang and S. L. Wong. "Functional Characterization and Localization of a Bacillus Subtilis Sortase and Its Substrate and Use of This Sortase System to Covalently Anchor a Heterologous Protein to the B. Subtilis Cell Wall for Surface Display." J Bacteriol 194, no. 1 (2012): 161-75. [http://www.ncbi.nlm.nih.gov/pubmed/22020651 PubMed]

Oussenko, I. A., R. Sanchez and D. H. Bechhofer. "Bacillus Subtilis Yhcr, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure." J Bacteriol 186, no. 16 (2004): 5376-83. [http://www.ncbi.nlm.nih.gov/pubmed/15292138 PubMed]