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

本项目的目标是开发实验方法来设计合成新的操纵子，用于细菌中多个基因的协调表达。具体目标是：(1)开发一种双基因表达系统，该系统允许方便地插入发夹、RNase切割位点和基因；(2)设计用于稳定操纵子的一个或多个编码区的合成发夹；(3)确认合成插入物形成RNA折叠算法预测的结构；(4)检测发夹强度、核糖核酸内切酶切割位点和操纵子基因位置对mRNA稳定性和蛋白质产生的影响；(5)检测基因中的RNase E位点和发夹中的RNase III位点对这些基因转录本稳定性的影响；(6)利用双酶代谢途径检测合成的操纵子，该途径具有易于测定的中间体。

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.