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Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome
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| Zeitschriftentitel: | Journal of Bacteriology |
|---|---|
| Personen und Körperschaften: | , |
| In: | Journal of Bacteriology, 176, 1994, 6, S. 1761-1763 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
|
| Schlagwörter: |
| author_facet |
Steinmetz, M Richter, R Steinmetz, M Richter, R |
|---|---|
| author |
Steinmetz, M Richter, R |
| spellingShingle |
Steinmetz, M Richter, R Journal of Bacteriology Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome Molecular Biology Microbiology |
| author_sort |
steinmetz, m |
| spelling |
Steinmetz, M Richter, R 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.176.6.1761-1763.1994 <jats:p>Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.</jats:p> Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome Journal of Bacteriology |
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1994 |
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Journal of Bacteriology |
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49 |
| title |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_unstemmed |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_full |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_fullStr |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_full_unstemmed |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_short |
Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_sort |
easy cloning of mini-tn10 insertions from the bacillus subtilis chromosome |
| topic |
Molecular Biology Microbiology |
| url |
http://dx.doi.org/10.1128/jb.176.6.1761-1763.1994 |
| publishDate |
1994 |
| physical |
1761-1763 |
| description |
<jats:p>Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.</jats:p> |
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| description | <jats:p>Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.</jats:p> |
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| imprint | American Society for Microbiology, 1994 |
| imprint_str_mv | American Society for Microbiology, 1994 |
| institution | DE-D275, DE-Bn3, DE-Brt1, DE-Zwi2, DE-D161, DE-Gla1, DE-Zi4, DE-15, DE-Pl11, DE-Rs1, DE-105, DE-14, DE-Ch1, DE-L229 |
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| spelling | Steinmetz, M Richter, R 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.176.6.1761-1763.1994 <jats:p>Delivery vectors for mini-Tn10 transposons function in Bacillus subtilis (M. A. Petit, C. Bruand, L. Janniére, and S. D. Ehrlich, J. Bacteriol. 172:6736-6740, 1990). Using this system, we identified a new gene (sytA) whose inactivation affected regulation of genes of sucrose metabolism. For cloning the sytA::Tn10 insertion in Escherichia coli, we developed a methodology similar to that commonly used for B. subtilis Tn917 insertions. We constructed a plasmid which can be used to insert (by in vivo recombination) a ColE1 origin linked to a spectinomycin resistance gene (ori-spc element) into mini-Tn10 transposons inserted into the B. subtilis chromosome. DNA extracted from a sytA::Tn10::ori-spc transformant was cut with restriction enzymes that do not cut into the Tn10::ori-spc sequence; plasmids containing the sytA::Tn10 insertion were cloned by self-ligation, followed by transformation of E. coli. To obtain the wild-type sytA region, one of these plasmids was ligated with an E. coli-B. subtilis shuttle vector conferring erythromycin resistance, and the hybrid was used to transform the wild-type B. subtilis strain. Erythromycin-resistant transformants, detected as spectinomycin sensitive, resulted from conversion of the insertion mutation by the resident wild-type locus. The shuttle plasmid containing the wild-type locus could then be recovered in E. coli.</jats:p> Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome Journal of Bacteriology |
| spellingShingle | Steinmetz, M, Richter, R, Journal of Bacteriology, Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome, Molecular Biology, Microbiology |
| title | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_full | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_fullStr | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_full_unstemmed | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_short | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| title_sort | easy cloning of mini-tn10 insertions from the bacillus subtilis chromosome |
| title_unstemmed | Easy cloning of mini-Tn10 insertions from the Bacillus subtilis chromosome |
| topic | Molecular Biology, Microbiology |
| url | http://dx.doi.org/10.1128/jb.176.6.1761-1763.1994 |