Using reporter genes and the Escherichia coli ASKA overexpression library in screens for regulators of the Gram negative envelope stress response.

We describe methods for screening the E. coliASKA overexpression library for clones that lead to altered expression of reporter genes. First, a promoter of interest is cloned upstream of either the lacZor luxCDABEgenes to yield reporter genes in which transcription is proportional to the levels of β-galactosidase or luminescence produced by strains carrying the reporter. The ASKA library is then condensed into two 96-well plates resulting in mixed preparations of 12 plasmids in each well. The plasmids in each well are transformed into the reporter strain and transformants are screened for either altered β-galactosidase or light production. The genes contained in ASKA clones that result in altered reporter gene expression are amplified and sequenced and the ASKA clone for the gene identified is retransformed into the parent reporter strain to confirm the effect. We have used screens like this one to look for new E. coligenes that, when over-expressed, result in the altered expression of promoters that are regulated by the envelope stress response. The identity of the clones can yield information about the nature of inducing cues and/or additional regulatory molecules. The techniques are broadly applicable to any microbial function of interest.