Coding

Part:BBa_K1351010

Designed by: Mona Dotzler   Group: iGEM14_LMU-Munich   (2014-10-05)

Cell wall-anchoring domain of B. subtilis endonuclease YhcR

Cell wall-anchoring domain (amino acids 1086 to 1217) of Bacillus subtilis endonuclease YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]). YhcR is secreted and covalently anchored to the petidoglycan wall by the sortase YhcS.

A YhcR-based surface display system has been shown to covalently anchor heterologous proteins to the B. subtilis cell wall (Liew et. al., 2012). It was used in the [http://2014.igem.org/Team:LMU-Munich/Project/Bakillus BaKillus project] to display pathogen-specific peptides and mediate adhesion of B. subtlis to pathogens.

It consists of four components: the sortase substrate with a signal peptide (BBa_K1351008), a linker (BBa_K1351009) and a cell wall-anchoring domain (this part) as well as the sortase YhcS itself (BBa_K1351011), whose overexpression increases the efficiency of the surface display.


This part was generated in a modified version of RFC25, where a strong Shine Dalgarno Sequence (SD) is included, and has the following prefix and suffix:

prefix with EcoRI, NotI, XbaI, SD and NgoMIV: GAATTCGCGGCCGCTTCTAGAGTAAGGAGGAGCCGGC
suffix with AgeI, SpeI, NotI and PstI: ACCGGTTAATACTAGTAGCGGCCGCTGCAG

Sites of restriction enzymes generating compatible overhangs have the same color:

EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red, NgoMIV and AgeI in orange. Shine-Dalgarno sequence and stop codons are underlined.


Sequence and Features


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


[edit]
Categories
//cds
//chassis/prokaryote/bsubtilis
Parameters
None