ArticleApplied and environmental microbiology2013
An improved method for oriT-directed cloning and functionalization of large bacterial genomic regions.
Article in Applied and environmental microbiology, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Identifying phenotypic and genetic traits for assessing pathogenic potential and biocontrol capacity in Burkholderia sensu lato strains.The ISME journal · 2026Article
- Direct cloning and heterologous expression of natural product biosynthetic gene clusters by transformation-associated recombination.Methods in enzymology · 2019Article
- Simple and rapid direct cloning and heterologous expression of natural product biosynthetic gene cluster in Bacillus subtilis via Red/ET recombineering.Scientific reports · 2016Article
- "Cre/loxP plus BAC": a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens.Scientific reports · 2016Article
- Mob/oriT, a mobilizable site-specific recombination system for unmarked genetic manipulation in Bacillus thuringiensis and Bacillus cereus.Microbial cell factories · 2016Article
- Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns.Synthetic and systems biotechnology · 2016Review
- Article
- Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2).Scientific reports · 2015Article
- Genome engineering and direct cloning of antibiotic gene clusters via phage ϕBT1 integrase-mediated site-specific recombination in Streptomyces.Scientific reports · 2015Article
- Tn5/7-lux: a versatile tool for the identification and capture of promoters in gram-negative bacteria.BMC microbiology · 2015Article
- Multicopy integration of mini-Tn7 transposons into selected chromosomal sites of a Salmonella vaccine strain.Microbial biotechnology · 2015Article
- Mining the metabiome: identifying novel natural products from microbial communities.Chemistry & biology · 2014Review
- Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A.Proceedings of the National Academy of Sciences of the United States of America · 2014Article
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Authors and funding
3 authors.
Funding
Abstract
We have made significant improvements to a broad-host-range system for the cloning and manipulation of large bacterial genomic regions based on site-specific recombination between directly repeated oriT sites during conjugation. Using two suicide capture vectors carrying flanking homology regions, oriT sites are recombined on either side of the target region. Using a broad-host-range conjugation helper plasmid, the region between the oriT sites is conjugated into an Escherichia coli recipient strain, where it is circularized and maintained as a chimeric mini-F vector. The cloned target region is functionalized in multiple ways to accommodate downstream manipulation. The target region is flanked with Gateway attB sites for recombination into other vectors and by rare 18-bp I-SceI restriction sites for subcloning. The Tn7-functionalized target can also be inserted at a naturally occurring chromosomal attTn7 site(s) or maintained as a broad-host-range plasmid for complementation or heterologous expression studies. We have used the oriTn7 capture technique to clone and complement Burkholderia pseudomallei genomic regions up to 140 kb in size and have created isogenic Burkholderia strains with various combinations of genomic islands. We believe this system will greatly aid the cloning and genetic analysis of genomic islands, biosynthetic gene clusters, and large open reading frames.
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