Protocol to Identify the Core Gene Supported by an Essential Gene in E. coli Bacteria Using a Genome-Wide Suppressor Screen

Authors

Isao Masuda, Thomas Jefferson UniversityFollow
Ya-Ming Hou, Thomas Jefferson UniversityFollow

Document Type

Protocol

Publication Date

3-29-2023

Comments

This article is the author's final published version in STAR Protocols, Volume 4, Issue 2, March 2023, Article number 102196.

The published version is available at https://doi.org/10.1016/j.xpro.2023.102196.

Copyright © 2023The Authors. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abstract

We describe here a genome-wide screening approach to identify the most critical core reaction among a network of many that are supported by an essential gene to establish cell viability. We describe steps for maintenance plasmid construction, knockout cell construction, and phenotype validation. We then detail isolation of suppressors, whole-genome sequencing analysis, and reconstruction of CRISPR mutants. We focus on E. coli trmD, which encodes an essential methyl transferase that synthesizes m1G37 on the 3'-side of the tRNA anticodon. For complete details on the use and execution of this protocol, please refer to Masuda et al. (2022).

Recommended Citation

Masuda, Isao and Hou, Ya-Ming, "Protocol to Identify the Core Gene Supported by an Essential Gene in E. coli Bacteria Using a Genome-Wide Suppressor Screen" (2023). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 236.
https://jdc.jefferson.edu/bmpfp/236

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

PubMed ID

36995932

Language

English