The Transcriptional Regulator MphR(A): Characterization and Circuit Engineering

Abstract

The MphR(A) transcriptional repressor protein was incorporated into high-efficiency ex-pression vectors, purified and characterized. Initial screens for crystallography solvents were conducted along with preliminary gel-shift and intrinsic fluorescence quenching ex-periments to obtain the equilibrium dissociation constant for the protein-DNA interac-tions. The constant was found to be 0.5 - 1.9 μM, depending on the method used. The gene was also incorporated into positive feedback circuits to detect macrolide antibiotics using various reporter genes and plasmid constructs. Qualitatively, the circuits showed a change in output upon the addition of MphR to the system.