Reporter Gene Repression Assay to Study Translational Regulation of a Target Gene

In This Article

Abstract

Source: Katz, N., et al. An Assay for Quantifying Protein-RNA Binding in Bacteria. J. Vis. Exp. (2019)

This video demonstrates an in vivo assay in bacteria to study the interaction of RNA-binding proteins (RBPs) with RNA. The bacterial cells are transformed with two plasmid constructs — a binding-site plasmid encoding an mRNA containing a fluorescent reporter gene downstream of an RBP-binding site — while an RBP plasmid expresses the RBP proteins under the control of an inducer. Upon inducer-mediated increase in RBP production, the RBPs bind to the mRNA, resulting in translational repression of the reporter via inhibition of ribosome binding.

Protocol

1. System Preparation

Design of binding-site plasmids
1. Design the binding site cassette as depicted in Figure 1. Each minigene contains the following parts (5' to 3'): Eagl restriction site, ∼40 bases of the 5' end of the kanamycin (Kan) resistance gene, pLac-Ara promoter, ribosome binding site (RBS), AUG of the mCherry gene, a spacer (δ), an RBP binding site, 80 bases of the 5' end of the mCherry gene, and an ApaLI restriction site.
  NOTE: To increase the success rate of the assay, design three binding-site cassettes for each binding site, with spacers consist....

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Results

Genetic circuit diagram showing cloning, transformation, and ribosome binding site regulation.
Figure 1: Overview of system design and cloning steps. Illustration of the cassette design for the binding site plasmid (left) and RBP-mCerulean plasmid (right). The next step is consecutive transformations of both plasmids into competent E. coli cel.......