| Summary: | 碩士 === 國立臺北科技大學 === 化學工程所 === 93 === In this research, we use linear transformation that takes advantage of the homologous sequence between two ends of linear DNA and the host cell E.coli, to transfer the target genes directly into the chromosome of the strain. Because this method enables expressing recombinant proteins continuously without plasmid, the problem of plasmid instability is solved.
In practice, we use ZSC114, a multi-auxotrophic E.coli strain that cannot metabolize glucose, mannose and lactose, as our host cell. First, we unite each of the three metabolism-related genes with lacZ gene, to prepare for a functional linear DNA. Then we put these linear substrates into the chromosome of ZSC114. We can make up for the auxotrophic phenotype of the original strain, and with expression of more lacZ genes, enhance the production of recombinant protein β-galactosidase.
Protein gel electrophoresis andβ-galactosidase activity assay are executed to measure the production of recombinant protein LacZ among our newly constructed strains. We find out that the production and activity of the genetic product increase while the copy number of lacZ gene rises in the bacterial chromosome.
We use the method of serial dilution culture to investigate genetic stability between the three-lacZ gene-carrying strain, ZSCGMtZ, and the plasmid expression system, ZSC114/pJL. We find out that in serial dilution culture, the ZSC114/pJL system would loss LacZ expression at 35th hour, but new strain ZSCGMtZ still keep quite high recombinant protein expression after 60 hours. The result indicates the genetic stability of new strain is better.
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