Development of a Plasmid Selection System for E. coli Host Based on Bacterial fab Genes

Background and Aim : Over the past few years, plasmid DNA (pDNA) vectors have played a crucial role in preventing infectious diseases and meeting the needs of researchers by facilitating gene transfer to prokaryotic and eukaryotic cells, whether bacterial or animals. Furthermore, progress towards all form of non-viral gene therapy has led exponential growth in the demand for safe plasmid vectors. As the goals for preparation of intact plasmid DNA have been increased over time, the attention of the health organizations and biotechnology research scientists is focused on antibiotic resistance genes in pDNA backbones as selectable markers which cause several issues including arising in multidrug-resistance (MDR) bacteria, probable responses of the host's immune system, and epigenetic changes. In order to proper handling of these problems, developing antibiotic-free selection systems such as overexpression of fabI gene can be convenient. Methods : To test the method in E. coli, fabI and fabV genes were used as the plasmid selectable markers in pcDNA۳.۱(+) vector which lacked the resistance related genes to Kanamycin and ampicillin. The recombinant strains were grown in adverse conditions with biocide Triclosan as the selective agent Results : The recomibant vector with fabI as a selective marker gene, enabled selection by Triclosan at ۶ µg/ml and the one with fabV selectable marker, was grown in selective media containing ۵۰۰ µg/ml Triclosan. However, plasmid stability and plasmid yield decreased for both recombinant vectors through plasmid extraction.Conclusion : While a variety of non-antibiotic selectable markers have been designated, the fabV-triclosan selection system provides a suitable plasmid choosing without using antibiotics resistance selectable markers.

کلیدواژه ها:

plasmid DNA (pDNA) vectors ، antibiotic-free selection systems ، The overexpression of fabI gene ، fabV gene