Abstract
The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem.
Highlights
Universal cloning vectors offer a series of advantages to facilitate their manipulation, which include their small size, presence of multiple cloning sites, selection markers, and high copy number
In this work we describe a new target sequence for IS10 transposition present in the commonly used pSU and pSW families of cloning vectors
Transposon Tn10 is not present in the originally sequenced genome of E. coli, IS10 element is present in 16% of E. coli and Shigella genomes in GenBank [27]
Summary
Universal cloning vectors offer a series of advantages to facilitate their manipulation, which include their small size, presence of multiple cloning sites, selection markers, and high copy number. The primarily used cloning vectors are associated with the pMB1 replicon They carry most often the beta-lactamase gene from Tn3, originally present in the historic vector pBR322 (GeneBank Accession number J01749; [1]), allowing plasmid selection by resistance to ampicillin. Cloning vectors based on the p15A replicon are the common alternative to pMB1-based vectors They have a moderate copy number and they are a better option when potentially toxic genes are to be cloned. Most of these vectors encode the cat gene from Tn9, as in original vector pACYC184 (GeneBank Accession number X06403; [2]), conferring chloramphenicol resistance (CmR). The most widely used family of p15A-derived cloning vectors is the pSU8 family [3, 4], which was designed to allow coexistence with a pMB1-ApR vector in the PLOS ONE | DOI:10.1371/journal.pone.0138615 September 16, 2015
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