Engineering mRNA Stability for Coordinated Expression of Multiple Genes in New Operons

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

The goal of this project is to develop the experimental methodologies to engineer synthetic novel operons for coordinated expression of multiple genes in bacteria. The specific aims are: (1) to develop a dual-gene expression system that allows for convenient insertion of hairpins, RNase cleavage sites, and genes; (2) to design synthetic hairpins to be used to stabilize one or more coding regions of an operon; (3) to confirm that the synthetic inserts form the structures predicted by RNA folding algorithms; (4) to test the effects of hairpin strength and endoribonuclease cleavage site location, and gene location in an operon on mRNA stability and protein production; (5) to test the effect of RNase E sites in the genes and RNase III sites in the hairpins on the stability of the transcript for these genes; and (6) to test the synthetic operon with a dual-enzyme metabolic pathway that has an easily assayable intermediate.