A cloning vector employing a versatile β-glucosidase as an indicator for recombinant clones

A mutant glucosidase, cpGluT, with activity toward chromogenic substrates (X-gal [5-bromo-4-chloro-3-idolyl-β-d-galactoside] and indican) and a fluorogenic 4-methylumbeliferyl-β-d-glucopyranoside (MUG) was constructed by replacing the monomeric β-glucosidase region (E314–N326) with designed multiple...

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Bibliographic Details
Published in:Analytical biochemistry 2012-06, Vol.425 (2), p.166-168
Main Authors: Cheong, Dea-Eun, Chang, Woo-Suk, Kim, Geun-Joong
Format: Article
Language:English
Subjects:
beta-glucosidase
clones
Cloning vector
fluorescence
Galactosides - chemistry
Galactosides - genetics
Galactosides - metabolism
genetic vectors
Genetic Vectors - genetics
Genetic Vectors - metabolism
hosts
Indican
Indican - chemistry
Indicator
Indoles - chemistry
MUG
mutants
ortho-Aminobenzoates - chemistry
phenotype
plasmids
Plasmids - analysis
Plasmids - metabolism
screening
Uridine Diphosphate Glucose - analogs & derivatives
Uridine Diphosphate Glucose - chemistry
X-gal
β-Glucosidase
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Summary:A mutant glucosidase, cpGluT, with activity toward chromogenic substrates (X-gal [5-bromo-4-chloro-3-idolyl-β-d-galactoside] and indican) and a fluorogenic 4-methylumbeliferyl-β-d-glucopyranoside (MUG) was constructed by replacing the monomeric β-glucosidase region (E314–N326) with designed multiple cloning sites. When expressed in hosts (lacZ+ and lacZ–), a vector containing the cpGluT produced a colored or fluorescent phenotype according to the substrate supplemented on LB plates without any inducer. cpGluT is readily incorporable into customized vectors and does not require special hosts to detect recombinant plasmids, thereby making screening recombinants more effective and less expensive.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.03.004