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; …
Offenlegungen
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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Diesen Artikel zitieren
Reporter Gene Repression Assay to Study Translational Regulation of a Target Gene. J. Vis. Exp. (Pending Publication), e21435, doi: (2023).