I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

ABSTRACT: We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expres

SUBMITTER: Szentes S 

PROVIDER: S-EPMC7049694 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature