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Title 

Generating in vivo cloning vectors for parallel cloning of large gene clusters by homologous recombination

Authors 

J LeeEugene RhaSoo-Jin YeomDae-Hee LeeEui Sung ChoiSeung Goo Lee

Publisher 

Public Library of Science

Issue Date 

2013

Citation 

Plos One, vol. 8, no. 11, pp. e79979-e79979

Abstract 

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 × 104 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.

ISSN 

1932-6203

Link 

http://dx.doi.org/10.1371/journal.pone.0079979

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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