LacZ selection of recombinant colonies

목차

1. Introduction
2. Materials
3. Protocol 1
4. Protocol 2
5. References

본문내용

1. Introduction

(1) α-Complementation
The E. coli enzyme β-galactosidase is a homo-tetramer of the protein product of the lacZ gene. Certain mutations in the 5' region of lacZ prevent subunit association. Because monomers lack enzyme activity, the failure to assemble leads to a Lac- phenotype. Such lacZ mutants are said to be subject to "α-complementation."
Obviously, α-complementation requires special host strains carrying an "α-complementable" lacZ mutation such as lacZΔM15. E. coli DH5α is such a strain.

(2) X-GAL ( 5-bromo-4-chloro-3-indolyl-beta-D-galactoside)
E. coli encodes a β-galactosidase that hydrolyzes the disaccharide lactose into the monosaccharides glucose and galactose. The activity of the enzyme can be assayed with a chromogenic substrate such as X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), which is converted by β-galactosiedsase into an insoluble dense blue compound.

참고 자료

J. Sambrook and D. W. Russell, 2001, Molecular Cloning A Laboratory manual Third Edition, COLD SPRING HARBOR LABORATORY PRESS, P 1.123 ~ 1.125, P 1.149~1.150
http://bio.classes.ucsc.edu/bio20L/content/molbio2/alphacomp.html