Format

Send to

Choose Destination
Plasmid. 2016 Jul;86:1-6. doi: 10.1016/j.plasmid.2016.05.001. Epub 2016 May 24.

New high-cloning-efficiency vectors for complementation studies and recombinant protein overproduction in Escherichia coli and Salmonella enterica.

Author information

1
Department of Microbiology, University of, 120 Cedar Street, Athens, GA 306022605 Georgia.
2
Department of Microbiology, University of, 120 Cedar Street, Athens, GA 306022605 Georgia. Electronic address: jescala@uga.edu.

Abstract

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 H6-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 H6-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.

KEYWORDS:

Complementation vectors; Escherichia coli; High-efficiency cloning vectors; Overexpression vectors; S. enterica; Type IIS restriction cloning

PMID:
27234933
PMCID:
PMC5126758
DOI:
10.1016/j.plasmid.2016.05.001
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center