Cloning:Article Title: Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity
Article Snippet: .. The following plasmids were used in this work: cloning vector pBluescript KS (Ap r ) (Stratagene), expression vector pET30a (Km r ) (Invitrogen), mobilizable broad-host-range promoter-probe vector pCM132 (Km r , ori RK2, lacZ reporter gene fusion vector), and helper plasmid pRK2013 ( 22 ). ..
Article Title: Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus
Article Snippet: .. When required, appropriate antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains V. parahaemolyticus WP1 Clinical isolate 17 AQ3354 Clinical isolate 17 TNB1 AQ3354, pvsA disrupted; Cm r This study TNB2 AQ3354, pvsD disrupted; Cm r This study TNB3 AQ3354, pvsE disrupted; Cm r This study TNB4 AQ3354, pvuB disrupted; Cm r This study E. coli DH5α endA1 hsdr17 (r K − m K + ) supE44 thi-1 recA1 gyrA96 relA1 Δ( argF-lacZYA ) U169 deoR [φ80d lac Δ( lacZ )M15)]; general cloning host Promega JM109 recA1 endA1 gylA96 thi hsdR17 supE44 relA1 Δ( lac-proAB )/F′ ( traD36 proAB + lacI q lac ZΔM15); general cloning host 72 SY327λ pir Δ( lac pro ) argE (Am) recA56 gyrA rpoB λ pir , host for π-requiring plasmids 35 SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu, λ pir; Km r ; host for π-requiring plasmids; conjugal donor 35 Plasmids pUC19 High-copy-number cloning vector; Ap r 61 pBluescript II KS(+) High-copy-number cloning vector; Ap r Stratagene pMW118 Low-copy-number cloning vector; Ap r Nippon Gene pACYC184 Low-copy-number cloning vector; Tc r Cm r 8 pKTN701 R6K- ori suicide vector for gene replacement; Cm r 39 pVP3151 Initially isolated FURTA-positive clone; pUC19 containing chromosomal 3,151-bp Pst I fragment from WP1; Ap r 17 pVS1 pMW118 containing chromosomal 3.2-kb Sal I- Sal I fragment from WP1; Ap r This study pVS1-1 pBluescript II KS(+) containing 373-bp Pst I- Kpn I fragment from pVS1; Ap r This study pVS2 pMW118 containing chromosomal 7.5-kb Hin dIII- Hin dIII fragment from WP1; Ap r This study pVPV2995 pBluescript II KS(+) containing the chromosomal 2,995-bp Eco RI- Sac I fragment from WP1; Ap r 17 pVFRD1 pACYC184 containing chromosomal 7.7-kb Sal I- Hin dIII fragment from WP1; Cm r This study pTNB1 pKTN701 containing 682-bp Xba I- Eco RI fragment within pvsA from pVS2; Cm r This study pTNB2 pKTN701 containing 748-bp Sac I- Kpn I fragment PCR amplified with primers PVSD-Kpn and PVSD-Sac with pVS2 as a template; Cm r This study pTNB3 pKTN701 containing 688-bp Kpn I- Eco RI fragment PCR amplified with primers PVSE-Kpn and PVSE-Eco with pVS2 as a template; Cm r This study pTNB4 pKTN701 containing 601-bp Sac I- Kpn I fragment PCR amplified with primers PVUB-Sac and PVUB-Kpn with pVFRD1 as a template; Cm r This study Open in a separate window Strains and plasmids used in this study .. Chromosomal DNAs of V. parahaemolyticus strains were extracted from overnight cultures with a Wizard genomic DNA purification kit (Promega), and plasmid DNA was routinely prepared with a plasmid miniprep kit (Bio-Rad), according to the manufacturer's protocols.
Expressing:Article Title: Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity
Article Snippet: .. The following plasmids were used in this work: cloning vector pBluescript KS (Ap r ) (Stratagene), expression vector pET30a (Km r ) (Invitrogen), mobilizable broad-host-range promoter-probe vector pCM132 (Km r , ori RK2, lacZ reporter gene fusion vector), and helper plasmid pRK2013 ( 22 ). ..
Article Title: Regulation of the Transposase of Tn 4652 by the Transposon-Encoded Protein TnpC
Article Snippet: .. P. putida was electrotransformed according to the protocol described by Sharma and Schimke ( 29 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference E. coli TG1 supE hsd Δ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λ c I ts 857 ind 1 S am7 nin 5 lac UV5-T7 gene 1 ) 32 P. putida PaW85 Tn 4652 4 PRS2000 Tn 4652 free 34 Plasmids a pBluescript KS Cloning vector (Ap r ) Stratagene pET19b Protein expression vector (Ap r ) Stratagene pET19-tnpA tnpA is fused with histidine tag in pET19b This work pKT240 Cloning vector (Ap r Km r ) 3 pEST1354 Plasmid containing Tn 4652 upstream of the pheBA operon 17 pKTtnpA(D/H) Tn 4652 tnpA gene within the 3.2-kb Dra I- Hin dIII fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) Tn 4652 tnpA and tnpC genes within the 3.7-kb Dra I- Pvu I fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) * pKTtnpA(D/P) with the tnpC gene disrupted by frameshift This work pKTGC/tnpA pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene This work pKTGC/tnpAC pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene with tnpC This work pGUS102 Promoter probe vector containing the gusA gene cloned as a 1.8-kb Eco RI fragment into pBR322 A. Eriksson pKTGUS Vector for translational fusions containing the gusA gene without translation initiation codon ATG in pKT240; Hin dIII restriction site designed at the 5′ end of the gusA is suitable for in-frame cloning This work pTr1 PDEL2-GC promoter region plus 42 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr2 PDEL2-GC promoter region plus 546 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr3 PDEL2-GC promoter region plus 1,166 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pKT-ACG gusA is cloned downstream of tnpC in pKTtnpA(D/P) This work (Fig. B) pKT-AdelCG Cla I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) pKT-a1CG Dra I- Cla I deletion derivative of pKT-ACG This work (Fig. B) pKT-a2CG Dra I- Nru I deletion derivative of pKT-ACG This work (Fig. B) pKT-CG Dra I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) Open in a separate window a Oligonucleotides used for construction of the plasmids are described in Materials and Methods. ..
Plasmid Preparation:Article Title: Molecular Characterization of a Novel Temperate Sinorhizobium Bacteriophage, ФLM21, Encoding DNA Methyltransferase with CcrM-Like Specificity
Article Snippet: .. The following plasmids were used in this work: cloning vector pBluescript KS (Ap r ) (Stratagene), expression vector pET30a (Km r ) (Invitrogen), mobilizable broad-host-range promoter-probe vector pCM132 (Km r , ori RK2, lacZ reporter gene fusion vector), and helper plasmid pRK2013 ( 22 ). ..
Article Title: Regulation of the Transposase of Tn 4652 by the Transposon-Encoded Protein TnpC
Article Snippet: .. P. putida was electrotransformed according to the protocol described by Sharma and Schimke ( 29 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference E. coli TG1 supE hsd Δ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λ c I ts 857 ind 1 S am7 nin 5 lac UV5-T7 gene 1 ) 32 P. putida PaW85 Tn 4652 4 PRS2000 Tn 4652 free 34 Plasmids a pBluescript KS Cloning vector (Ap r ) Stratagene pET19b Protein expression vector (Ap r ) Stratagene pET19-tnpA tnpA is fused with histidine tag in pET19b This work pKT240 Cloning vector (Ap r Km r ) 3 pEST1354 Plasmid containing Tn 4652 upstream of the pheBA operon 17 pKTtnpA(D/H) Tn 4652 tnpA gene within the 3.2-kb Dra I- Hin dIII fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) Tn 4652 tnpA and tnpC genes within the 3.7-kb Dra I- Pvu I fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) * pKTtnpA(D/P) with the tnpC gene disrupted by frameshift This work pKTGC/tnpA pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene This work pKTGC/tnpAC pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene with tnpC This work pGUS102 Promoter probe vector containing the gusA gene cloned as a 1.8-kb Eco RI fragment into pBR322 A. Eriksson pKTGUS Vector for translational fusions containing the gusA gene without translation initiation codon ATG in pKT240; Hin dIII restriction site designed at the 5′ end of the gusA is suitable for in-frame cloning This work pTr1 PDEL2-GC promoter region plus 42 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr2 PDEL2-GC promoter region plus 546 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr3 PDEL2-GC promoter region plus 1,166 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pKT-ACG gusA is cloned downstream of tnpC in pKTtnpA(D/P) This work (Fig. B) pKT-AdelCG Cla I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) pKT-a1CG Dra I- Cla I deletion derivative of pKT-ACG This work (Fig. B) pKT-a2CG Dra I- Nru I deletion derivative of pKT-ACG This work (Fig. B) pKT-CG Dra I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) Open in a separate window a Oligonucleotides used for construction of the plasmids are described in Materials and Methods. ..
Article Title: Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus
Article Snippet: .. When required, appropriate antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains V. parahaemolyticus WP1 Clinical isolate 17 AQ3354 Clinical isolate 17 TNB1 AQ3354, pvsA disrupted; Cm r This study TNB2 AQ3354, pvsD disrupted; Cm r This study TNB3 AQ3354, pvsE disrupted; Cm r This study TNB4 AQ3354, pvuB disrupted; Cm r This study E. coli DH5α endA1 hsdr17 (r K − m K + ) supE44 thi-1 recA1 gyrA96 relA1 Δ( argF-lacZYA ) U169 deoR [φ80d lac Δ( lacZ )M15)]; general cloning host Promega JM109 recA1 endA1 gylA96 thi hsdR17 supE44 relA1 Δ( lac-proAB )/F′ ( traD36 proAB + lacI q lac ZΔM15); general cloning host 72 SY327λ pir Δ( lac pro ) argE (Am) recA56 gyrA rpoB λ pir , host for π-requiring plasmids 35 SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu, λ pir; Km r ; host for π-requiring plasmids; conjugal donor 35 Plasmids pUC19 High-copy-number cloning vector; Ap r 61 pBluescript II KS(+) High-copy-number cloning vector; Ap r Stratagene pMW118 Low-copy-number cloning vector; Ap r Nippon Gene pACYC184 Low-copy-number cloning vector; Tc r Cm r 8 pKTN701 R6K- ori suicide vector for gene replacement; Cm r 39 pVP3151 Initially isolated FURTA-positive clone; pUC19 containing chromosomal 3,151-bp Pst I fragment from WP1; Ap r 17 pVS1 pMW118 containing chromosomal 3.2-kb Sal I- Sal I fragment from WP1; Ap r This study pVS1-1 pBluescript II KS(+) containing 373-bp Pst I- Kpn I fragment from pVS1; Ap r This study pVS2 pMW118 containing chromosomal 7.5-kb Hin dIII- Hin dIII fragment from WP1; Ap r This study pVPV2995 pBluescript II KS(+) containing the chromosomal 2,995-bp Eco RI- Sac I fragment from WP1; Ap r 17 pVFRD1 pACYC184 containing chromosomal 7.7-kb Sal I- Hin dIII fragment from WP1; Cm r This study pTNB1 pKTN701 containing 682-bp Xba I- Eco RI fragment within pvsA from pVS2; Cm r This study pTNB2 pKTN701 containing 748-bp Sac I- Kpn I fragment PCR amplified with primers PVSD-Kpn and PVSD-Sac with pVS2 as a template; Cm r This study pTNB3 pKTN701 containing 688-bp Kpn I- Eco RI fragment PCR amplified with primers PVSE-Kpn and PVSE-Eco with pVS2 as a template; Cm r This study pTNB4 pKTN701 containing 601-bp Sac I- Kpn I fragment PCR amplified with primers PVUB-Sac and PVUB-Kpn with pVFRD1 as a template; Cm r This study Open in a separate window Strains and plasmids used in this study .. Chromosomal DNAs of V. parahaemolyticus strains were extracted from overnight cultures with a Wizard genomic DNA purification kit (Promega), and plasmid DNA was routinely prepared with a plasmid miniprep kit (Bio-Rad), according to the manufacturer's protocols.
Clone Assay:Article Title: Regulation of the Transposase of Tn 4652 by the Transposon-Encoded Protein TnpC
Article Snippet: .. P. putida was electrotransformed according to the protocol described by Sharma and Schimke ( 29 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference E. coli TG1 supE hsd Δ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λ c I ts 857 ind 1 S am7 nin 5 lac UV5-T7 gene 1 ) 32 P. putida PaW85 Tn 4652 4 PRS2000 Tn 4652 free 34 Plasmids a pBluescript KS Cloning vector (Ap r ) Stratagene pET19b Protein expression vector (Ap r ) Stratagene pET19-tnpA tnpA is fused with histidine tag in pET19b This work pKT240 Cloning vector (Ap r Km r ) 3 pEST1354 Plasmid containing Tn 4652 upstream of the pheBA operon 17 pKTtnpA(D/H) Tn 4652 tnpA gene within the 3.2-kb Dra I- Hin dIII fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) Tn 4652 tnpA and tnpC genes within the 3.7-kb Dra I- Pvu I fragment cloned into pKT240 This work (Fig. ) pKTtnpA(D/P) * pKTtnpA(D/P) with the tnpC gene disrupted by frameshift This work pKTGC/tnpA pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene This work pKTGC/tnpAC pKT240 plus pDEL2-GC promoter region plus the promoterless tnpA gene with tnpC This work pGUS102 Promoter probe vector containing the gusA gene cloned as a 1.8-kb Eco RI fragment into pBR322 A. Eriksson pKTGUS Vector for translational fusions containing the gusA gene without translation initiation codon ATG in pKT240; Hin dIII restriction site designed at the 5′ end of the gusA is suitable for in-frame cloning This work pTr1 PDEL2-GC promoter region plus 42 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr2 PDEL2-GC promoter region plus 546 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pTr3 PDEL2-GC promoter region plus 1,166 nt of the coding region of tnpA fused with gusA in pKTGUS This work (Fig. B) pKT-ACG gusA is cloned downstream of tnpC in pKTtnpA(D/P) This work (Fig. B) pKT-AdelCG Cla I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) pKT-a1CG Dra I- Cla I deletion derivative of pKT-ACG This work (Fig. B) pKT-a2CG Dra I- Nru I deletion derivative of pKT-ACG This work (Fig. B) pKT-CG Dra I- Cfr 10I deletion derivative of pKT-ACG This work (Fig. B) Open in a separate window a Oligonucleotides used for construction of the plasmids are described in Materials and Methods. ..
other:Article Title: A Vibrio vulnificus Type IV Pilin Contributes to Biofilm Formation, Adherence to Epithelial Cells, and Virulence
Article Snippet: Isopropyl -β- d -thiogalactopyranoside (IPTG) was used at a final concentration of 0.2 or 1 mM. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strains, vector, or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus C7184/OP Wild type (clinical isolate) 73 C7184DΩ C7184 with disrupted pilD This laboratory C7184AΩ C7184 with disrupted pilA This study MO6-24/OP Wild type (clinical isolate) 34 PAC1 Wild type (environmental isolate) M. Coyle P. aeruginosa PAK-NP Nonpiliated mutant 46 PAK-NP(pRP383) Nonpiliated mutant expressing PilA from V. vulnificus This study E. coli DH5α supE44lacU169 (Δ lacZ ΔM15) hsdR17 recA lendA1gyrA96 thi-1 relA1 BRL SM10-λpir thi thr leu tonAlacY supE recA [RP4-2-Tc::Mu] λpir R6K Km r 51 BL21 F − ompT hsd S B (r B − m B − ) gal dcm 61 , 62 pFLAG-MAC Ap r FLAG fusion cloning vector Sigma pFLAG-ATS Ap r FLAG fusion cloning vector Sigma Cloning vectors pZErO-2 Kan r Zero background cloning vector Invitrogen pMMB67EH.cam Cm r Ap r broad-host-range cloning vector, lacI q / tac promoter 17 pBluescript II KS + Ap r phagemid cloning vector Stratagene pEP185.2 Cm r suicide vector 22 Recombinant plasmids pRP-P38 9- to 10-kb PstI fragment from C7184 carrying the pilABCD cluster cloned into pZErO-2 This study pRP102 2.04-kb PCR-generated fragment encompassing pilA cloned into pKS + This study pRP102Ω 2.04-kb PCR-generated fragment encompassing pilA, disrupted with the 2.0-kb Ω interposon, cloned into pEP185.2 This study pRP383 624-bp XhoI-HindIII fragment encompassing pilA cloned into pMMB67EH.cam This study pRPA110 PCR-generated pilA cloned into pFLAG-MAC This study pRPA111 PCR-generated pilA cloned into pFLAG-ATS This study Open in a separate window Bacterial strains and plasmids used in this study DNA manipulations.
Article Title: Effects of Combination of Different -10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ? S -Dependent Transcription in Pseudomonas putida
Article Snippet: Antibiotics were added at the following final concentrations: ampicillin at 100 μg/ml for E. coli ; carbenicillin at 1,500 μg/ml, kanamycin at 50 μg/ml, and tetracycline at 10 μg/ml for P. putida . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or construction Source or reference Strains E. coli TG1 supE hsdΔ5 thi Δ( lac-proAB ) F′ ( traD36 proAB + lacI q lacZ ΔM15) 6 BL21(DE3) hsdS gal (λcI ts 857 ind 1 Sam 7 nin 5 lac UV5-T7 gene 1 39 S17-1 λpir Tp r Sm r recA thi pro (r − m + ) RP4::2 Tc::Mu::Km Tn 7 λpir 30 C118 λpir Δ( ara-leu ) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 λpir phage lysogen 16 P. putida PaW85 Tn 4652 3 PKS54 Tn 4652 rpoS ::Km r This work PKSRpoS P. putida PKS54 rpoS under control of Ptac promoter and lacI q repressor; Tc r Plasmids a pBluescript KS(+) Cloning vector (Ap r ) Stratagene pBlcrpoS-I pBluescript KS(+) containing 1-kb PCR-amplified rpoS cloned into Eco RV site pUC4K Cloning vector containing Km r gene from transposon Tn 903 (Ap r Km r ) 34 , 46 pBlcrpoS-Km r 2.5-kb rpoS -Km r sequence containing Xba I- Eco RI fragment from pUC4K cloned into Eco 72I site in rpoS gene This work pUTmini.Tn 5 luxAB Delivery plasmid for mini-Tn 5 luxAB (Ap r Tet r ) 8 pUTrpoS-Km r 2.5-kb rpoS -Km r sequence-containing Xba I- Eco RI fragment from pBlcrpoS-Km r cloned into pUTmini-Tn 5 luxAB This work pBlcrpoS-II pBluescript KS(+) containing 900-bp PCR-amplified rpoS cloned into Eco RV site This work pET24d Protein expression vector (Km r ) Stratagene pET24d-rpoS pET24d containing rpoS gene from pBlcrpoS-II inserted into Nco I and Hin dIII sites This work pKTlacZ Cloning vector (Ap r ) 18 pBRlacItac Ptac promoter and lacI q repressor in 2.2-kb Nru I- Eco RI fragment from plasmid pMMB208 cloned into Eco RV- Eco RI-cleaved pBR322 — b pMir61 rpoS gene of P. putida KT2440 in pUN19Ø 36 pBRlacItac-rpoS rpoS in 1-kb Xho I- Hin dIII fragment from plasmid pMir61 cloned into Sal I- Sma I-cleaved pBRlacItac This work pUC18Not pUC18 with Not I restriction site in multicloning region (Km r ) 16 pUCptac-rpoS 3.2-kb rpoS expression cassette Ptac-rpoS-lacI q from pBRlacItac-rpoS inserted into Bam HI- Kpn I-cleaved pUC18Not This work pUTptac-rpoS Ptac-rpoS-lacI q from pUCptac-rpoS inserted into Not I-cleaved pUTmini-Tn 5 luxAB This work Open in a separate window a The strategy of cloning of the fusion promoters and their mutant forms into promoter probe vector pKTlacZ is shown in Materials and Methods and in Fig. and . b —, Hõrak and Kivisaar, submitted.
Isolation:Article Title: Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus
Article Snippet: .. When required, appropriate antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains V. parahaemolyticus WP1 Clinical isolate 17 AQ3354 Clinical isolate 17 TNB1 AQ3354, pvsA disrupted; Cm r This study TNB2 AQ3354, pvsD disrupted; Cm r This study TNB3 AQ3354, pvsE disrupted; Cm r This study TNB4 AQ3354, pvuB disrupted; Cm r This study E. coli DH5α endA1 hsdr17 (r K − m K + ) supE44 thi-1 recA1 gyrA96 relA1 Δ( argF-lacZYA ) U169 deoR [φ80d lac Δ( lacZ )M15)]; general cloning host Promega JM109 recA1 endA1 gylA96 thi hsdR17 supE44 relA1 Δ( lac-proAB )/F′ ( traD36 proAB + lacI q lac ZΔM15); general cloning host 72 SY327λ pir Δ( lac pro ) argE (Am) recA56 gyrA rpoB λ pir , host for π-requiring plasmids 35 SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu, λ pir; Km r ; host for π-requiring plasmids; conjugal donor 35 Plasmids pUC19 High-copy-number cloning vector; Ap r 61 pBluescript II KS(+) High-copy-number cloning vector; Ap r Stratagene pMW118 Low-copy-number cloning vector; Ap r Nippon Gene pACYC184 Low-copy-number cloning vector; Tc r Cm r 8 pKTN701 R6K- ori suicide vector for gene replacement; Cm r 39 pVP3151 Initially isolated FURTA-positive clone; pUC19 containing chromosomal 3,151-bp Pst I fragment from WP1; Ap r 17 pVS1 pMW118 containing chromosomal 3.2-kb Sal I- Sal I fragment from WP1; Ap r This study pVS1-1 pBluescript II KS(+) containing 373-bp Pst I- Kpn I fragment from pVS1; Ap r This study pVS2 pMW118 containing chromosomal 7.5-kb Hin dIII- Hin dIII fragment from WP1; Ap r This study pVPV2995 pBluescript II KS(+) containing the chromosomal 2,995-bp Eco RI- Sac I fragment from WP1; Ap r 17 pVFRD1 pACYC184 containing chromosomal 7.7-kb Sal I- Hin dIII fragment from WP1; Cm r This study pTNB1 pKTN701 containing 682-bp Xba I- Eco RI fragment within pvsA from pVS2; Cm r This study pTNB2 pKTN701 containing 748-bp Sac I- Kpn I fragment PCR amplified with primers PVSD-Kpn and PVSD-Sac with pVS2 as a template; Cm r This study pTNB3 pKTN701 containing 688-bp Kpn I- Eco RI fragment PCR amplified with primers PVSE-Kpn and PVSE-Eco with pVS2 as a template; Cm r This study pTNB4 pKTN701 containing 601-bp Sac I- Kpn I fragment PCR amplified with primers PVUB-Sac and PVUB-Kpn with pVFRD1 as a template; Cm r This study Open in a separate window Strains and plasmids used in this study .. Chromosomal DNAs of V. parahaemolyticus strains were extracted from overnight cultures with a Wizard genomic DNA purification kit (Promega), and plasmid DNA was routinely prepared with a plasmid miniprep kit (Bio-Rad), according to the manufacturer's protocols.
Polymerase Chain Reaction:Article Title: Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus
Article Snippet: .. When required, appropriate antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains V. parahaemolyticus WP1 Clinical isolate 17 AQ3354 Clinical isolate 17 TNB1 AQ3354, pvsA disrupted; Cm r This study TNB2 AQ3354, pvsD disrupted; Cm r This study TNB3 AQ3354, pvsE disrupted; Cm r This study TNB4 AQ3354, pvuB disrupted; Cm r This study E. coli DH5α endA1 hsdr17 (r K − m K + ) supE44 thi-1 recA1 gyrA96 relA1 Δ( argF-lacZYA ) U169 deoR [φ80d lac Δ( lacZ )M15)]; general cloning host Promega JM109 recA1 endA1 gylA96 thi hsdR17 supE44 relA1 Δ( lac-proAB )/F′ ( traD36 proAB + lacI q lac ZΔM15); general cloning host 72 SY327λ pir Δ( lac pro ) argE (Am) recA56 gyrA rpoB λ pir , host for π-requiring plasmids 35 SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu, λ pir; Km r ; host for π-requiring plasmids; conjugal donor 35 Plasmids pUC19 High-copy-number cloning vector; Ap r 61 pBluescript II KS(+) High-copy-number cloning vector; Ap r Stratagene pMW118 Low-copy-number cloning vector; Ap r Nippon Gene pACYC184 Low-copy-number cloning vector; Tc r Cm r 8 pKTN701 R6K- ori suicide vector for gene replacement; Cm r 39 pVP3151 Initially isolated FURTA-positive clone; pUC19 containing chromosomal 3,151-bp Pst I fragment from WP1; Ap r 17 pVS1 pMW118 containing chromosomal 3.2-kb Sal I- Sal I fragment from WP1; Ap r This study pVS1-1 pBluescript II KS(+) containing 373-bp Pst I- Kpn I fragment from pVS1; Ap r This study pVS2 pMW118 containing chromosomal 7.5-kb Hin dIII- Hin dIII fragment from WP1; Ap r This study pVPV2995 pBluescript II KS(+) containing the chromosomal 2,995-bp Eco RI- Sac I fragment from WP1; Ap r 17 pVFRD1 pACYC184 containing chromosomal 7.7-kb Sal I- Hin dIII fragment from WP1; Cm r This study pTNB1 pKTN701 containing 682-bp Xba I- Eco RI fragment within pvsA from pVS2; Cm r This study pTNB2 pKTN701 containing 748-bp Sac I- Kpn I fragment PCR amplified with primers PVSD-Kpn and PVSD-Sac with pVS2 as a template; Cm r This study pTNB3 pKTN701 containing 688-bp Kpn I- Eco RI fragment PCR amplified with primers PVSE-Kpn and PVSE-Eco with pVS2 as a template; Cm r This study pTNB4 pKTN701 containing 601-bp Sac I- Kpn I fragment PCR amplified with primers PVUB-Sac and PVUB-Kpn with pVFRD1 as a template; Cm r This study Open in a separate window Strains and plasmids used in this study .. Chromosomal DNAs of V. parahaemolyticus strains were extracted from overnight cultures with a Wizard genomic DNA purification kit (Promega), and plasmid DNA was routinely prepared with a plasmid miniprep kit (Bio-Rad), according to the manufacturer's protocols.
Amplification:Article Title: Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus
Article Snippet: .. When required, appropriate antibiotics were added to the media at the following concentrations: ampicillin, 100 μg/ml; chloramphenicol, 10 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Description Reference or source Strains V. parahaemolyticus WP1 Clinical isolate 17 AQ3354 Clinical isolate 17 TNB1 AQ3354, pvsA disrupted; Cm r This study TNB2 AQ3354, pvsD disrupted; Cm r This study TNB3 AQ3354, pvsE disrupted; Cm r This study TNB4 AQ3354, pvuB disrupted; Cm r This study E. coli DH5α endA1 hsdr17 (r K − m K + ) supE44 thi-1 recA1 gyrA96 relA1 Δ( argF-lacZYA ) U169 deoR [φ80d lac Δ( lacZ )M15)]; general cloning host Promega JM109 recA1 endA1 gylA96 thi hsdR17 supE44 relA1 Δ( lac-proAB )/F′ ( traD36 proAB + lacI q lac ZΔM15); general cloning host 72 SY327λ pir Δ( lac pro ) argE (Am) recA56 gyrA rpoB λ pir , host for π-requiring plasmids 35 SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu, λ pir; Km r ; host for π-requiring plasmids; conjugal donor 35 Plasmids pUC19 High-copy-number cloning vector; Ap r 61 pBluescript II KS(+) High-copy-number cloning vector; Ap r Stratagene pMW118 Low-copy-number cloning vector; Ap r Nippon Gene pACYC184 Low-copy-number cloning vector; Tc r Cm r 8 pKTN701 R6K- ori suicide vector for gene replacement; Cm r 39 pVP3151 Initially isolated FURTA-positive clone; pUC19 containing chromosomal 3,151-bp Pst I fragment from WP1; Ap r 17 pVS1 pMW118 containing chromosomal 3.2-kb Sal I- Sal I fragment from WP1; Ap r This study pVS1-1 pBluescript II KS(+) containing 373-bp Pst I- Kpn I fragment from pVS1; Ap r This study pVS2 pMW118 containing chromosomal 7.5-kb Hin dIII- Hin dIII fragment from WP1; Ap r This study pVPV2995 pBluescript II KS(+) containing the chromosomal 2,995-bp Eco RI- Sac I fragment from WP1; Ap r 17 pVFRD1 pACYC184 containing chromosomal 7.7-kb Sal I- Hin dIII fragment from WP1; Cm r This study pTNB1 pKTN701 containing 682-bp Xba I- Eco RI fragment within pvsA from pVS2; Cm r This study pTNB2 pKTN701 containing 748-bp Sac I- Kpn I fragment PCR amplified with primers PVSD-Kpn and PVSD-Sac with pVS2 as a template; Cm r This study pTNB3 pKTN701 containing 688-bp Kpn I- Eco RI fragment PCR amplified with primers PVSE-Kpn and PVSE-Eco with pVS2 as a template; Cm r This study pTNB4 pKTN701 containing 601-bp Sac I- Kpn I fragment PCR amplified with primers PVUB-Sac and PVUB-Kpn with pVFRD1 as a template; Cm r This study Open in a separate window Strains and plasmids used in this study .. Chromosomal DNAs of V. parahaemolyticus strains were extracted from overnight cultures with a Wizard genomic DNA purification kit (Promega), and plasmid DNA was routinely prepared with a plasmid miniprep kit (Bio-Rad), according to the manufacturer's protocols.
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