Chen W, Kallio P T, Bailey J E
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Gene. 1993 Aug 16;130(1):15-22. doi: 10.1016/0378-1119(93)90341-y.
A novel cross-regulation system employing two pairs of interacting promoter-repressor systems was constructed using the tac-lacI and lambda pL-cI promoter-operator-repressor systems. In particular, transcription of the cat gene and the fused cI gene is regulated by the tac promoter, while transcription of the lacI gene is controlled by the lambda pL promoter. In order to compare CAT production from this new system with a currently employed transcription control configuration, a control expression vector utilizing the constitutive repressor synthesis configuration was also constructed. In this construct, cat is under the control of the tac promoter, and lac repressor is provided from a single copy of the lacIq allele included in the plasmid. Induction results using different copy number vectors indicate that induced cat expression levels are at least twofold higher using the cross-regulation system which has very low basal expression. These results match well with previous mathematical modeling predictions indicating excellent control of basal expression and also higher cloned-gene expression post-induction over a broad range of copy numbers for a cross-regulation control configuration. Induction of the cross-regulation system both up-regulated the activation pathway and down-regulated the inhibition pathway, shifting the system steady-state from lac repressor expression to cat and cI expression. The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.
利用tac-lacI和λ pL-cI启动子-操纵子-阻遏物系统构建了一种采用两对相互作用的启动子-阻遏物系统的新型交叉调控系统。具体而言，cat基因和融合的cI基因的转录受tac启动子调控，而lacI基因的转录由λ pL启动子控制。为了将该新系统的CAT产量与当前使用的转录控制配置进行比较，还构建了一种利用组成型阻遏物合成配置的对照表达载体。在该构建体中，cat受tac启动子控制，而lac阻遏物由质粒中包含的单拷贝lacIq等位基因提供。使用不同拷贝数载体的诱导结果表明，使用具有非常低基础表达的交叉调控系统时，诱导的cat表达水平至少高出两倍。这些结果与先前的数学建模预测非常吻合，表明对于交叉调控控制配置，在广泛的拷贝数范围内，基础表达得到了出色的控制，诱导后克隆基因的表达也更高。交叉调控系统的诱导既上调了激活途径，又下调了抑制途径，将系统稳态从lac阻遏物表达转变为cat和cI表达。本文提出的控制策略应同样适用于调控不同宿主中的转录。
