Preparation of Genomic Overexpression Library

Abstract

This protocol is used to identify/select for Escherichia coli genes that, when overexpressed in Vibrio cholerae Hsp33 (hslO gene) deletion mutant, protect against oxidative heat stress and, by extension, against HOCl-mediated protein damage. In the referenced publication, I found that V. cholerae mutant lacking Hsp33 (in addition to its established role in HOCl stress) has severe temperature sensitive phenotypes on MacConkey plates (this phenotype is not found in E. coli ΔhslO mutant). I constructed genomic expression libraries from E. coli and transformed the libraries into V. cholerae ΔhslO mutant. I then searched for complementing E. coli proteins that rescue the temperature sensitive phenotype of V. cholerae ΔhslO mutant on MacConkey plate. This protocol might work for searching complementation of other gene deletion mutants that have a good selective phenotype.