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

Published: June 29, 2023

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 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 restr…

Representative Results

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 cells, with RBP plasmids first. Double-transformants are then tested for their mCherry expression levels in increasing inducer concentrations; …

Disclosures

The authors have nothing to disclose.

Materials

Ampicillin sodium salt SIGMA A9518
Magnesium sulfate (MgSO4) ALFA AESAR 33337
48 plates Axygen P-5ML-48-C-S
8-lane plates Axygen RESMW8I
96-well plates Axygen P-DW-20-C
96-well plates for plate reader Perkin Elmer 6005029
ApaLI NEB R0507
Binding site sequences Gen9 Inc. and Twist Bioscience see Table 1
E. coli TOP10 cells Invitrogen C404006
Eagl-HF NEB R3505
Glycerol BIO LAB 71205
Incubator TECAN Liconic incubator
Kanamycin solfate SIGMA K4000
KpnI- HF NEB R0142
Ligase NEB B0202S
Liquid-handling robotic system TECAN EVO 100, MCA 96-channel
MATLAB analysis software Mathworks
Multi-pipette 8 lanes Axygen BR703710
N-butanoyl-L-homoserine lactone (C4-HSL) Cayman K40982552 019
PBS buffer Biological Industries 020235A
Plate reader TECAN Infinite F200 PRO
Q5 HotStart Polymerase NEB M0493
RBP sequences Addgene 27121 & 40650 see Table 2
Sodium Chloride (NaCl) BIO LAB 190305
SV Gel and PCR Clean-Up System Promega A9281
Tryptone BD 211705

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Cite This Article
Reporter Gene Repression Assay to Study Translational Regulation of a Target Gene. J. Vis. Exp. (Pending Publication), e21435, doi: (2023).

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