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

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 × 10(4) positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb

SUBMITTER: Lee J 

PROVIDER: S-EPMC3823602 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature