title: A highly efficient molecular cloning platform that utilises a small bacterial toxin gene.
abstract: Molecular cloning technologies that have emerged in recent years are more efficient and simpler to use than traditional strategies, but many have the disadvantages of requiring multiple steps and expensive proprietary enzymes. We have engineered cloning vectors containing variants of IbsC, a 19-residue toxin from Escherichia coli K-12. These toxic peptides offer selectivity to minimise the background, labour, and cost associated with conventional molecular cloning. As demonstrated with the cloning of reporter genes, this "detox cloning" system consistently produced over 95 % positive clones. Purification steps between digestion and ligation are not necessary, and the total time between digestion and plating of transformants can be as little as three hours. Thus, these IbsC-based cloning vectors are as reliable and amenable to high-throughput cloning as commercially available systems, and have the advantage of being more time-efficient and cost-effective.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton Ontario, L8S 4K1, Canada.
authors.forename: Wendy W K
authors.initials: WW
authors.lastname: Mok
authors.forename: Yingfu
authors.initials: Y
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D048168:Escherichia coli K12 / Q000737:chemistry / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods; D008969:Molecular Sequence Data
chemical_list: D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; C555654:IbsC protein, E coli