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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Autores principales:	Jeongmin Lee, Eugene Rha, Soo-Jin Yeom, Dae-Hee Lee, Eui-Sung Choi, Seung-Goo Lee
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7fb832802ccd47479ec1a2c710224629
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spelling	oai:doaj.org-article:7fb832802ccd47479ec1a2c7102246292021-11-18T08:47:22ZGenerating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.1932-620310.1371/journal.pone.0079979https://doaj.org/article/7fb832802ccd47479ec1a2c7102246292013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24244585/?tool=EBIhttps://doaj.org/toc/1932-6203A 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.Jeongmin LeeEugene RhaSoo-Jin YeomDae-Hee LeeEui-Sung ChoiSeung-Goo LeePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 11, p e79979 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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.
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.
format	article
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
title	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_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_sort	generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/7fb832802ccd47479ec1a2c710224629
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Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination.

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