Abstract
Directed enzyme evolution is now a routine approach to improve desirable biocatalytic properties. When only a low-throughput screen is available to detect improved variants from a mutant gene library, it is imperative that cloning efficiency be maximized during library synthesis to avoid wasting effort screening empty plasmids. To achieve this we developed pUCXKT, a gain-of-function positive selection expression vector. Insertion of genes amplified using a specialized downstream PCR primer restores key regulatory and genetic elements necessary for co-expression of a kanamycin resistance marker adjacent to the pUCXKT cloning region. We show that pUCXKT enables 100 % cloning efficiency as well as high-level expression of inserted genes. Unlike previous positive selection expression plasmids, the strategy we used to design pUCXKT is readily adaptable to different vector backbones, antibiotic marker genes, and multiple cloning regions.
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Acknowledgments
This research was supported by Genesis Oncology Trust (contract GOT-1252-RPG) and Marsden Research Fund (contract number VUW0901) grants to DFA. GAP and EMW were supported by Tertiary Education Commission Top Achiever Doctoral Scholarships. EMW received salary support from the Cancer Society of New Zealand (contract 13/01).
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Gareth A Prosser and Elsie M Williams contributed equally to this work.
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Prosser, G.A., Williams, E.M., Sissons, J.A. et al. A gain-of-function positive-selection expression plasmid that enables high-efficiency cloning. Biotechnol Lett 37, 383–389 (2015). https://doi.org/10.1007/s10529-014-1673-4
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DOI: https://doi.org/10.1007/s10529-014-1673-4