| I have developed a new tool: the ability to affect and coordinate multiple gene expression by directing the processing and stabilities of mRNA from novel operons. By itself or in combination with the other methods of gene expression control, mRNA processing should be a powerful tool to control the expression of several genes simultaneously.; An expression system that contains the araBAD promoter was developed to allow for the easy introduction of mRNA stability control elements into various regions of a dual-gene operon through DNA cassettes. The effects of endoribonuclease sites, secondary structures 5′ and 3′ of the coding regions, and gene placement on protein production and mRNA stability and steady-state levels were tested in a dual-gene operon containing two reporter genes, gfp and lacZ. Depending on the combination of control elements used the relative steady-state transcript and protein levels encoded by the two reporter genes could be changed up to 100-fold and 1,000-fold, respectively.; Identical expression systems were developed in both high- and low-copy plasmids to separately test the effects of DNA copy number and mRNA steady-state levels on relative protein production from a dual-gene operon.; Identical expression systems were developed under both native arabinose-dependent and engineered arabinose-independent transporter protein (AraE) promoter control. Differences in cell populations, enzyme assays, and trends in relative production were examined in the two cell lines harboring identical dual-gene operons. Cells harboring the engineered araE had homogeneous cell populations across all inducer concentrations and a more linear response of protein production to changes in inducer concentration. Cells harboring the native araE had dual cell populations, exhibiting “all-or-none” phenomena, and a sigmoidal response of protein production to changes in inducer concentrations.; Finally, the directed mRNA processing and stability technology was applied to a metabolic pathway of interest, the carotenoid pathway. The flux through this pathway was successfully controlled to a variety of different cases, using the previously developed mRNA stability control element systems. In addition, intermediates that were not normally seen were also detected in the pathway. |
