| Summary: | 碩士 === 逢甲大學 === 化學工程學所 === 92 === In response to a massive production of recombinant proteins, the physiological state in Escherichia coli is forced to alter. This will usually lead to the retardation of cell growth commonly referred to stress responses. In particular, the target proteins are subject to ptroteolytic attack as a result of the heat-shock response.
In this study, an enormous production of LacZ was chosen a model to study the potential problems caused by the stress response. To achieve this goal, the encoding gene was placed under the control of the T7 promoter on a multiple-copy-number plasmid. Meanwhile, the substitute of the T7 promoter with the tac promoter was made to regulate the expression of LacZ. It was found that the production of LacZ directed by the T7 promoter was prone to degradation upon with IPTG exceeding 100 �嵱. However, the LacZ instability was absent when expressed by the tac promoter even with the full dosage of IPTG. Obviously, this could be attributed to the outcome of the protein overproduction-incurred stress. To further examine the underlying mechanism, a series of vectors was developed to contain promoter-free reporter genes along with phage attachment sites. The latter was aimed at insertion of the plasmid DNA into bacterial chromosome via the phage-mediated integration. Consequently, it would allow monitoring the expression of genes of interest as long as the DNAs carrying their promoter were fused to the reporter gene including lacZ, xylE, and gfp on the plasmid.
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