Protocol to identify the core gene supported by an essential gene in E. coli bacteria using a genome-wide suppressor screen

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).1 [Display omitted] •Removing the essential gene while maintaining a temporary state of cell viability•Maintaining the temporary cell viability by expressing the gene in a temperature control•High-temperature inactivation of the gene allows genome-wide screening of suppressors•Mapping suppressor mutations identifies the core gene at the limit of cell viability Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.