Plasmid Preparation:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Bioassay:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Mutagenesis:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Cloning:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Polymerase Chain Reaction:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Construct:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Isolation:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
Starch:Article Title: The Paralogous Pairs of Genes Involved in Clavulanic Acid and Clavam Metabolite Biosynthesis Are Differently Regulated in Streptomyces clavuligerus
Article Snippet: .. Bacterial strains and plasmids used in this study Strain, plasmid, or cosmid Descriptiona Reference or source Bacillus sp. strain ATCC 27860 Indicator strain for alanyl clavam bioassay 34 K. pneumoniae ATCC 15380 Indicator strain for clavulanic acid bioassay 35 S. clavuligerus NRRL3585 Wild type National Regional Research Laboratory (NRRL) S. clavuligerus ccaR::tsrA ccaR deletion mutant; ccaR replaced by tsr; Tsrr 2 S. clavuligerus C1G Contains the egfp reporter gene fused to the ceaS1 promoter; integrated at the C31 attB site in the chromosome This study S. clavuligerus C2G Contains the egfp reporter gene fused to the ceaS2 promoter; integrated at the C31 attB site in the chromosome This study Plasmids and cosmids K6L2 pLAFR3-based cosmid clone spanning the clavulanic acid gene cluster 1 p2.8-18 pUC18 containing the 5 end of ceaS1 and upstream region 42 p5.7 pUC118 containing blsI, pahI, and flanking sequences 42 pBB5.3A pUC119 containing the 5.3-kb BamHI/BglII fragment encompassing pcbR and ceaS2 from pTZ40; Ampr Jensen et al., unpublished pCR2.1TOPO E. coli phagemid cloning vector for rapid cloning of PCR fragments; Ampr Kanr Invitrogen pIJ8660 Promoterless EGFP reporter plasmid; free living in E. coli and integrates into the Streptomyces chromosome; Aprr 41 pIJ8660-ceaS2 EGFP reporter construct; pIJ8660 containing the 707-bp BamHI/KpnI fragment from pTOPO-ceaS2-8 encompassing the ceaS2 promoter region fused to the egfp gene This study pSET152 E. coli cloning vector; integrates into the Streptomyces chromosome; Aprr NRRL pSET-ceaS1 EGFP reporter construct; pSET152 containing the 2.37-kb BamHI/EcoRI fragment from pTO6-ceaS1 encompassing the ceaS1 promoter fused to the egfp gene This study pTO6 pIJ8660 with aac(3)IV replaced by aadA; Spcr 28 pTO6-ceaS1 EGFP reporter construct; pTO6 containing the 777-bp BamHI/KpnI fragment from pTOPO-ceaS1-4 This study pTOPO-cea52-8 pCR2.1TOPO containing a 721-bp PCR fragment encompassing the ceaS2 promoter region This study pTOPO-ceaS1-4 pCR2.1TOPO containing a 784-bp PCR fragment encompassing the ceaS1 promoter region This study pTZ40 pTZ18R containing an 26-kb BamHI/PstI fragment from cosmid K6L2 spanning the clavulanic acid gene cluster; Ampr Jensen et al., unpublished a Ampr, ampicillin resistance; Aprr, apramycin resistance; Kanr, kanamycin resistance, Spcr, spectinomycin resistance; Tsrr, thiostrepton resistance. on M arch 22, 2015 by G E O R G IA N C O U R T U N IV http://jb.asm .org/ D ow nloaded from erus cultures for the isolation of chromosomal DNA were grown in trypticase soy broth supplemented with 1% starch (TSBS), and cultures for the isolation of exconjugants were grown in AS-1 medium supplemented with 10 mM MgCl2, as described earlier (42). ..
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