Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase
Abstract Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the fi...
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Veröffentlicht in: | FEMS microbiology letters 1997-05, Vol.150 (1), p.49-54 |
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creator | Stoß, Oliver Mogk, Axel Schumann, Wolfgang |
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Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the β-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated. |
doi_str_mv | 10.1111/j.1574-6968.1997.tb10348.x |
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Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the β-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.</description><identifier>ISSN: 0378-1097</identifier><identifier>EISSN: 1574-6968</identifier><identifier>DOI: 10.1111/j.1574-6968.1997.tb10348.x</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>amyE ; Bacillus subtilis ; bgaB ; Cassettes ; cat‐86 ; Chloramphenicol ; Chloromycetin ; Chromosomes ; Codons ; Deoxyribonucleic acid ; DNA ; DNA biosynthesis ; DnaK protein ; E coli ; Galactosidase ; Gene expression ; Gene fusion ; Gene sequencing ; Heat ; Heat shock ; Integration ; Integration vector ; Microbiology ; Neomycin ; Plasmids ; Post-transcription ; Recombinant DNA ; Regulatory sequences ; Replication origins ; Reporter gene ; Thermal stability ; Translation ; α-Amylase ; β-Galactosidase</subject><ispartof>FEMS microbiology letters, 1997-05, Vol.150 (1), p.49-54</ispartof><rights>1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V. 1997</rights><rights>1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c2889-cca094f7628ee92c34e5cd4211fe0d00b25cb927b82b88b359350db859cb60143</citedby><cites>FETCH-LOGICAL-c2889-cca094f7628ee92c34e5cd4211fe0d00b25cb927b82b88b359350db859cb60143</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1111%2Fj.1574-6968.1997.tb10348.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1111%2Fj.1574-6968.1997.tb10348.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27901,27902,45550,45551</link.rule.ids></links><search><creatorcontrib>Stoß, Oliver</creatorcontrib><creatorcontrib>Mogk, Axel</creatorcontrib><creatorcontrib>Schumann, Wolfgang</creatorcontrib><title>Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase</title><title>FEMS microbiology letters</title><description>Abstract
Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the β-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.</description><subject>amyE</subject><subject>Bacillus subtilis</subject><subject>bgaB</subject><subject>Cassettes</subject><subject>cat‐86</subject><subject>Chloramphenicol</subject><subject>Chloromycetin</subject><subject>Chromosomes</subject><subject>Codons</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA biosynthesis</subject><subject>DnaK protein</subject><subject>E coli</subject><subject>Galactosidase</subject><subject>Gene expression</subject><subject>Gene fusion</subject><subject>Gene sequencing</subject><subject>Heat</subject><subject>Heat shock</subject><subject>Integration</subject><subject>Integration vector</subject><subject>Microbiology</subject><subject>Neomycin</subject><subject>Plasmids</subject><subject>Post-transcription</subject><subject>Recombinant DNA</subject><subject>Regulatory sequences</subject><subject>Replication origins</subject><subject>Reporter gene</subject><subject>Thermal stability</subject><subject>Translation</subject><subject>α-Amylase</subject><subject>β-Galactosidase</subject><issn>0378-1097</issn><issn>1574-6968</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1997</creationdate><recordtype>article</recordtype><sourceid>BENPR</sourceid><recordid>eNqVkc2OFCEUhYnRxHb0HYiuq-SnfsDEhTOZ0UnauNE1AepWTXUQSqBmpl_LlU_hM0nZE1e6kAS4yTnfvZCD0EtKalrW60NN276pOtmJmkrZ19lQwhtR3z9Cuz_SY7QjvBcVJbJ_ip6ldCCENIx0O_Tj2meYos7zLeBbsDlEPJZtg085rjbPfsKpHA4qG5YjzlH75Io_eO3wuKZSJGwg3wF4HGFaixjicSt_S2HE59rOzq0Jp9Xk2c0Jaz_gfAPYTPq8OJcQM0Q8gQcM3oZhm6rxDehcpayNA_zzezVpp8sD0zzoBM_Rk1G7BC8e7jP05ery88WHav_p_fXFu31lmRCyslYT2Yx9xwSAZJY30NqhYZSOQAZCDGutkaw3ghkhDG8lb8lgRCut6Qht-Bl6deq7xPBthZTVIayx_D0pxjkVhDPeF9ebk8vGkFKEUS1x_qrjUVGitqTUQW1xqC0OtSWlHpJS9wV-e4LvZgfH_yDV1cd9IwvfnviwLv-gq7_N_QXfG7FS</recordid><startdate>19970501</startdate><enddate>19970501</enddate><creator>Stoß, Oliver</creator><creator>Mogk, Axel</creator><creator>Schumann, Wolfgang</creator><general>Blackwell Publishing Ltd</general><general>Oxford University Press</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7QL</scope><scope>7T7</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>RC3</scope></search><sort><creationdate>19970501</creationdate><title>Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase</title><author>Stoß, Oliver ; Mogk, Axel ; Schumann, Wolfgang</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c2889-cca094f7628ee92c34e5cd4211fe0d00b25cb927b82b88b359350db859cb60143</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1997</creationdate><topic>amyE</topic><topic>Bacillus subtilis</topic><topic>bgaB</topic><topic>Cassettes</topic><topic>cat‐86</topic><topic>Chloramphenicol</topic><topic>Chloromycetin</topic><topic>Chromosomes</topic><topic>Codons</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA biosynthesis</topic><topic>DnaK protein</topic><topic>E coli</topic><topic>Galactosidase</topic><topic>Gene expression</topic><topic>Gene fusion</topic><topic>Gene sequencing</topic><topic>Heat</topic><topic>Heat shock</topic><topic>Integration</topic><topic>Integration vector</topic><topic>Microbiology</topic><topic>Neomycin</topic><topic>Plasmids</topic><topic>Post-transcription</topic><topic>Recombinant DNA</topic><topic>Regulatory sequences</topic><topic>Replication origins</topic><topic>Reporter gene</topic><topic>Thermal stability</topic><topic>Translation</topic><topic>α-Amylase</topic><topic>β-Galactosidase</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Stoß, Oliver</creatorcontrib><creatorcontrib>Mogk, Axel</creatorcontrib><creatorcontrib>Schumann, Wolfgang</creatorcontrib><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Genetics Abstracts</collection><jtitle>FEMS microbiology letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Stoß, Oliver</au><au>Mogk, Axel</au><au>Schumann, Wolfgang</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase</atitle><jtitle>FEMS microbiology letters</jtitle><date>1997-05-01</date><risdate>1997</risdate><volume>150</volume><issue>1</issue><spage>49</spage><epage>54</epage><pages>49-54</pages><issn>0378-1097</issn><eissn>1574-6968</eissn><abstract>Abstract
Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the β-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><doi>10.1111/j.1574-6968.1997.tb10348.x</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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source | Oxford University Press Journals All Titles (1996-Current); Wiley Online Library Journals Frontfile Complete; Alma/SFX Local Collection |
subjects | amyE Bacillus subtilis bgaB Cassettes cat‐86 Chloramphenicol Chloromycetin Chromosomes Codons Deoxyribonucleic acid DNA DNA biosynthesis DnaK protein E coli Galactosidase Gene expression Gene fusion Gene sequencing Heat Heat shock Integration Integration vector Microbiology Neomycin Plasmids Post-transcription Recombinant DNA Regulatory sequences Replication origins Reporter gene Thermal stability Translation α-Amylase β-Galactosidase |
title | Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable β-galactosidase |
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