Coding

Part:BBa_K1351007:Design

Designed by: Mona Dotzler   Group: iGEM14_LMU-Munich   (2014-10-05)
Revision as of 09:45, 5 October 2014 by Mdotzler (Talk | contribs) (→‎References)


CWB domain of B. subtilis minor autolysin LytE


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 459


Design Notes

Due to the N-terminal signal peptide, this part allows only C-terminal fusions of proteins to be displayed on the cell surface.

The CWB domain of LytE consists of three N‐terminal LysM cell wall‐binding motifs, ranging from amino acid 28 to 193. To minimize possible effects of a translational fusion to the CWB domain, the part includes the downstream unstructered region until amino acid 207. An additional linker can be inserted to allow more structural flexibilty.


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.

LytE_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC ATGAAAAAGCAAATCATTACAGCTACGACAGC

LytE_207_SNP_Age_rev: gatcCTGCAGcggccgctACTAGTattaACCGGT CGATGAAGATGAAGCAGGTTTTGAGC

A SpeI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers:

LytE_SpeI399mut_fwd: GTCCTGAAACTGAAAGGTTCAACcAGTTCAAGCAGCTCCAGCTCATCAAAAG

LytE_SpeI399mut_rev: CTTTTGATGAGCTGGAGCTGCTTGAACTgGTTGAACCTTTCAGTTTCAGGAC

References

Chen, C. L., S. C. Wu, W. M. Tjia, C. L. Wang, M. J. Lohka and S. L. Wong. "Development of a Lyte-Based High-Density Surface Display System in Bacillus Subtilis." Microb Biotechnol 1, no. 2 (2008): 177-90. [http://www.ncbi.nlm.nih.gov/pubmed/21261835 PubMed]