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 expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.

Gold-Nanostar-Based Surface-Enhanced Raman Scattering Substrates for Gastric Carcinoma Detection

“…Surface-enhanced Raman scattering (SERS) is a useful analytical technique for detecting trace levels of biochemical analytes. Because of strong plasmonic enhancement by noble-metal nanostructures, single-molecule identification has been demonstrated with SERS. − The current SERS , and other methods ,, of measurement are basically based on the reaction of 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-gal) with SA-β-gal to produce an insoluble blue substance as a marker for detection; however, this method has the problems of high cost and long staining time. In the construction of substrates, most of the substrates of other SERS methods used simple colloidal particles that showed lower uniformity and stability.…”

“…Because of strong plasmonic enhancement by noblemetal nanostructures, single-molecule identification has been demonstrated with SERS. 19−21 The current SERS 22,23 and other methods 12,24,25 substance as a marker for detection; however, this method has the problems of high cost and long staining time. In the construction of substrates, most of the substrates of other SERS methods used simple colloidal particles that showed lower uniformity and stability.…”