BBa_K1351011 1 BBa_K1351011 Bacillus subtilis sortase YhcS 2014-10-04T11:00:00Z 2015-06-01T12:55:40Z 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. YhcS_ENX_SD_Ngo_fwd: gatcGAATTCgcggccgctTCTAGAgtaaggaggaaGCCGGC GTGAAAAAAGTTATTCCACTATTCATCATTGC YhcS_SNP_rev: gatcCTGCAGcggccgctACTAGTa TTAAGTCACTCGTTTTCCATATATAATATAGCG An EcoRI site was removed by changing T to C at position 399 without altering the protein sequence by fusion PCR with the following mutagenesis primers: YhcS_EcoRI435mut_fwd: CGTCTTCTCCTGTCATATGGAGAATTtACATATGAAATTGTAAAAACAAAAATTGTCG YhcS_EcoRI435mut_rev: CGACAATTTTTGTTTTTACAATTTCATATGTaAATTCTCCATATGACAGGAGAAGACG ''Bacillus subtilis'' sortase YhcS (UniProt [http://www.uniprot.org/uniprot/P54603 P54603]), responsible for anchoring its substrae YhcR (UniProt [http://www.uniprot.org/uniprot/P54602 P54602]) covalently to the peptidoglycan wall of ''B. subtilis''. 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 (this part), a linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) and a cell wall-anchoring domain ([http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) as well as the sortase YhcS itself ([http://parts.igem.org/Part:BBa_K1351011 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: |<span style="color:blue">GAATTC</span><span style="color:green">GCGGCCGC</span>T<span style="color:red">TCTAGA</span>GT<u>AAGGAGG</u>A<span style="color:orange">GCCGGC</span> |- |suffix with AgeI, SpeI, NotI and PstI: |<span style="color:orange">ACCGGT</span><u>TAA</u>T<span style="color:red">AC<u>TAG</u><u>T</u></span><u>A</u><span style="color:green"><u>G</u>CGGCCGC</span><span style="color:blue">CTGCAG</span> |} Sites of restriction enzymes generating compatible overhangs have the same color: <span style="color:blue">EcoRI</span> and <span style="color:blue">PstI</span> in blue, <span style="color:green">NotI</span> in green, <span style="color:red">XbaI</span> and <span style="color:red">SpeI</span> in red, <span style="color:orange">NgoMIV</span> and <span style="color:orange">AgeI</span> in orange. Shine-Dalgarno sequence and stop codons are underlined. false false _1726_ 4206 20048 9 In stock false none false Mona Dotzler BBa_K1351011_sequence 1 gtgaaaaaagttattccactattcatcattgctgccggcctagtgatcgcaggctatggaggctttaaattgatcgatacgaatacgaaaacggaacagacattaaaagaagcaaagctggcggctaaaaagccgcaggaagcttcggggacgaaaaatagcacagatcaagcaaagaacaaagcatcatttaagcctgagaccggccaagcgagcggcattttagaaataccgaaaatcaatgcagagcttccgatcgtggagggcaccgatgctgatgatttagaaaaaggtgtcgggcattacaaggacagctattatcctgatgaaaacggacaaattgtgctgtcagggcatcgggataccgtgtttcgccggacgggagagctggaaaagggggatcagctccgtcttctcctgtcatatggagaatttacatatgaaattgtaaaaacaaaaattgtcgataaggatgacacatctattatcacgctccagcatgaaaaggaagagctgattctgacgacctgctatccgttttcatacgtcggaaatgccccgaagcgctatattatatatggaaaacgagtgacttaa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z