基因工程原理 第5章 柯斯质粒等其他高级载体.ppt

仅仅考虑用最强的启动子是不够的，还必须考虑过量表达对宿主细胞的影响。 把基因置于一个可调控启动子控制之下。 most of those in current use contain one of the following controllable promoters: λ PL, T7, trc (tac), or BAD. Table 5.3 shows the different levels of expression that can be achieved when the gene for chloramphenicol transacetylase (CAT) is placed under the control of three of these promoters. Control of expression of chloramphenicol acetyltransferase (CAT，氯霉素乙酰转移酶) in E. coli by three different promoters. The levels of CAT are expressed as μg/mg total protein. * Strategy for regulating the expression of genes cloned into a pET vector. The gene for T7 RNA polymerase (gene 1) is inserted into the chromosome of E. coli and transcribed from the lac promoter; therefore, it will be expressed only if the inducer IPTG is added. The T7 RNA polymerase will then transcribe the gene cloned into the pET vector. If the protein product of the cloned gene is toxic, it may be necessary to further reduce the transcription of the cloned gene before induction. The T7 lysozyme encoded by a compatible plasmid, pLysS, will bind to any residual T7 RNA polymerase made in the absence of induction and inactivate it. Also, the presence of lac operators between the T7 promoter and the cloned gene will further reduce transcription of the cloned gene in the absence of the inducer IPTG. * The λ PL promoter system combines very tight transcriptional control with high levels of gene expression. This is achieved by putting the cloned gene under the control of the PL promoter carried on a vector, while the PL promoter is controlled by a cI repressor gene in the E. coli host. This cI gene is itself under the control of the tryptophan (trp) promoter (Fig. 5.12). In the absence of exogenous tryptophan, the cI gene is transcribed and the cI repressor binds to the PL promoter, preventing expression of the cloned gene. Upon addition of tryptophan, the trp repressor binds to the cI gene, preventing synthesis of the cI repressor. In the absence of cI rep