Plasmid Preparation:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Bioassay:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Polymerase Chain Reaction:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Northern Blot:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Disruption:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Mutagenesis:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Cloning:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
Construct:Article Title: Two Sets of Paralogous Genes Encode the Enzymes Involved in the Early Stages of Clavulanic Acid and Clavam Metabolite Biosynthesis in Streptomyces clavuligerus
Article Snippet: .. All Streptomyces liquid cultures were grown at 28°C on a rotary shaker at 250 rpm. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or cosmid Description a Source reference Strains Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 36 E. coli ESS Indicator strain for cephamycin C bioassay A. L. Demain, Drew University, Madison, N.J. E. coli BW25113/pIJ790 Recombination host for Redirect PCR targeting system 16 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 37 S. clavuligerus NRRL3585 Wild type Northern Regional Research Laboratory, Peoria, Ill. S. clavuligerus 4B and 4B-C ceaS2 disruption mutant ( ceaS2 :: apr ) 20 S. clavuligerus O2FS ceaS2 frameshift mutant This study S. clavuligerus orf3 :: apr bls disruption mutant 20 S. lividans TK24 Plasmid-less cloning host (SLP2 − SLP3 − ), str-6 D. A. Hopwood, John Innes Institute, Norwich, United Kingdom Plasmids and cosmids 6G9 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a 14E10 pWE15-derived cosmid carrying DNA fragment encoding ceaS1 , bls1 , pah1 , and oat1 and flanking sequences 22a p2.8-18 pUC18 containing 2.8-kb Eco RI fragment carrying part of ceaS1 This study p5K-6 pUC118 containing 4.3-kb Nco I fragment carrying pah1 , oat1 , and upstream sequences but with an internal Kpn I fragment deleted to remove pah1 This study p5.7 pUC118 containing 5.7-kb Eco RI fragment carrying the 3′ end of ceaS1 , all of bls1 , pah1 , and oat1 , and flanking sequences This study p5.7-T p5.7 containing tsr from pTSR#8 inserted at Fse I site within bls1 This study p5.7-TH E. coli-Streptomyces shuttle vector formed by fusing p5.7T to pJOE829 This study pApOrf6 Disruption construct carrying oat2 disrupted with apr 20 pCAD2-3 pUC120 carrying ceaS2 and bls2 20 pCAD2-3(L1-5) pCAD2-3 but with a frameshift mutation in ceaS2 This study pCAD2-3(L1-5)486 E. coli-Streptomyces shuttle vector: pCAD2-3(L1-5) fused to pIJ486 This study pFDNeo-S pUC18 carrying neo from Tn 5 13 pIJ486 Streptomyces plasmid vector, Tsr r D. A. Hopwood pIJ773 Template plasmid for PCR targeting; acc ( 3 ) IV plus oriT 16 pJOE829 Streptomyces plasmid vector; Hyg r J. Altenbucher, University of Stuttgart, Stuttgart, Germany pNEO5K-6A p5K-6 containing neo from pFDNeo-S inserted at Rsr II site in oat1 This study pTSR#8 pUC118 containing tsr gene from pIJ702 1 pUC18 E. coli cloning vector; Amp r Stratagene pUC118 E. coli phagemid; Amp r 44 pUC120Apr pUC120 containing the apr gene flanked by Nco I sites 34 Open in a separate window a Hyg r , hygromycin resistance; Amp r , ampicillin resistance. ..
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