Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.
A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which w...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2013-01-01
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| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24244585/?tool=EBI |
| _version_ | 1818383670601318400 |
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| author | Jeongmin Lee Eugene Rha Soo-Jin Yeom Dae-Hee Lee Eui-Sung Choi Seung-Goo Lee |
| author_facet | Jeongmin Lee Eugene Rha Soo-Jin Yeom Dae-Hee Lee Eui-Sung Choi Seung-Goo Lee |
| author_sort | Jeongmin Lee |
| collection | DOAJ |
| description | A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10(4) positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells. |
| first_indexed | 2024-12-14T03:10:04Z |
| format | Article |
| id | doaj.art-7fb832802ccd47479ec1a2c710224629 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T03:10:04Z |
| publishDate | 2013-01-01 |
| publisher | Public Library of Science (PLoS) |
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| series | PLoS ONE |
| spelling | doaj.art-7fb832802ccd47479ec1a2c7102246292022-12-21T23:19:18ZengPublic Library of Science (PLoS)PLoS ONE1932-62032013-01-01811e7997910.1371/journal.pone.0079979Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.Jeongmin LeeEugene RhaSoo-Jin YeomDae-Hee LeeEui-Sung ChoiSeung-Goo LeeA robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10(4) positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24244585/?tool=EBI |
| spellingShingle | Jeongmin Lee Eugene Rha Soo-Jin Yeom Dae-Hee Lee Eui-Sung Choi Seung-Goo Lee Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. PLoS ONE |
| title | Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. |
| title_full | Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. |
| title_fullStr | Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. |
| title_full_unstemmed | Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. |
| title_short | Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination. |
| title_sort | generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24244585/?tool=EBI |
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