Journal
PLASMID
Volume 86, Issue -, Pages 1-6Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.plasmid.2016.05.001
Keywords
Type IIS restriction cloning; High-efficiency cloning vectors; Complementation vectors; Overexpression vectors; Escherichia coli; S. enterica
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Funding
- USPHS grant [R01 GM062203]
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Galloway et al. recently described a method to alter vectors to include Type IIS restriction enzymes for high efficiency cloning. Utilizing this method, the multiple cloning sites of complementation and overexpression vectors commonly used in our laboratory were altered to contain recognition sequences of the Type IIS restriction enzyme, BspQI. Use of this enzyme increased the rate of cloning success to >97% efficiency. L(+)-Arabinose-inducible complementation vectors and overexpression vectors encoding N-terminal recombinant tobacco etch virus protease (rTEV)-cleavable H-6-tags were altered to contain BspQI sites that allowed for cloning into all vectors using identical primer overhangs. Additionally, a vector used for directing the synthesis of proteins with a C-terminal, rTEV-cleavable H-6-tag was engineered to contain BspQI sites, albeit with different overhangs from that of the previously mentioned vectors. Here we apply a method used to engineer cloning vectors to contain BspQI sites and the use of each vector in either in vivo complementation studies or in vitro protein purifications. (C) 2016 Elsevier Inc. All rights reserved.
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