CmR This study pAL18 2133 bp fragment of approximately 1 kb upstream and 1 kb downstream of pilT cloned in XbaI and SalI site of pDM4. CmR This study Table 3 Primers used in this study Primer Primer sequence 5′-3′ RE site pilA LFF GAGCTCACGCGT-CTTACTTGCCGGATCATTACCAAC AZD6738 in vivo SphI pilA LFR CTGCAG-CCTTCTTTATAGTTTAGTTTAC PstI pilA RFF CTGCAGGTAGATAAACTAAGCCACTTTCATGTG PstI pilA RFR GGATCCGCATGCTCAAGGCTTCTGTCAATCTTGTTC MluI CAM PstIF GCCTGCAGGTAAGAGGTTCCAACTTTCAC PstI CAM PstIR TGATCTGCAGTTACGCCCCGCCCTGCCACTCATC PstI PilC-A GCATGTCCTAGGGTCAAGCTTAGATATTGCTGAA AvrII PilC-B TATATCGCATCGCCAAATAGCATATTTTTTATTCC
PilC-C GCTATTTGGCGATGCGATATAATATACTTTTAAAAA PilC-D GCATGTGTCGACGTCCTGAGAAAATATCTAGACA SalI PilT-A CATTATGTCGACTATGCAACAGTTCTTGATGGT SalI PilT-B TACTACAATGTATAGTAATTTTCTTATCATATCAAG PilT-C AGAAAATTACTATACATTGTAGTAAGGTAATCA PilT-D CATTATTCTAGACAGGATTAACGGCAGCTAAAA XbaI PilQ-A3 GCATGTCCTAGG TCAGTCAATGGAAGCACAGAT AvrII PilQ-B3 TATCTGCTATCATGTTAGAACAACTAATAACTTCTT PilQ-C3 TTGT TCTAACATGATAGCAGATAATAGTTGCAAA
PilQ-D3 GCATGTGTCGACAGAAAGTAATGTTGTTGGTATTT SalI RT-PCR primers PilA_A GATCCCGATGTACTCTAACTA PilA_B CCATTAGCTCAACTAGTGAGAA PilA_C ATCTTAGCAGCTGTAGCAATA PilA_D GGGGTAGTACTTTAAATCCT PilA_E CTTACTGAGTTACTTGTTGTTAT PilA_F GTCTTTCTGATCTATATGCTTC buy AZD4547 PilC_A GTCAAGCTTAGATATTGCTGAA PilC_B GTCTCTGGAGCACTGTTTGTAT PilC_C AAGGTAGTATTGATGCTGACAC PilC_D CCGTTGCTAAAGACACCATA PilC_E GATGCGATATAATATACTTTTAAAAA PilC_F CGAATTGGTATTGGCCAGAT PilQ_A TATGGTCAGGTAGAAGATGTAA PilQ_B CATCAATTTACCTTACTAATGTAT PilQ_C GCCTGAGCAGTAGTATAGTTT selleck chemicals llc PilQ_D AGTTGGTGCTGGAAAATCTAC PilQ_E CAGGATAGTTTCTTCTACTAAA PilT_A
CTATTAGGCGTGAAAGCAGTT PilT_B TAGTAATTTTCTTATCATATCAAG PilT_C ATGATGCGAGATTTAGGGTA PilT_D CAGCAGGTGGAAATACAGAT PilT_E TACATTGTAGTAAGGTAATCA PilT_F GGTAGAGTTGAATCAGCGTTTA Construction of deletion mutants of pilA, pilC, pilQ, and pilT in FSC237 Left and right flanking regions of pilA (FTT0890c, SCHU S4 nomenclature) were PCR amplified using the primer pairs pilA_LFF/pilA_LFR and pilA_RFF/pilA_RFR, and cloned into pGEMT-easy (Promega). The left flank was excised with EcoRI and PstI and the right flank was excised with BamHI and PstI. The fragments were ligated into an EcoRI/BamHI digested pBluescript KS+ vector (Stratagene), giving rise to pSMP47. A chloramphenicol resistance gene was PCR amplified from pDM4 with the primer pair CAM_PstIF/CAM_PstIR, digested with PstI, and cloned into 3-Methyladenine in vitro pSMP47, generating pSMP48 containing the left and right flanks of pilA disrupted by a chloramphenicol cassette. The mutated allele of pilA was excised from pSMP48 with SphI and MluI, cloned into pSMP22, and the resulting plasmid pSMP50CAM (Table 2) was introduced into strain FSC237 by conjugal mating as previously described .