| Summary: | 碩士 === 國立清華大學 === 生命科學系 === 90 === In this study, Cascade TM expression system was used to express the toxic protein, vaccinia virus topoisomerase I . Cascade TM system was regulated by two circuits to amplify gene expression levels. The expression system is tightly regulated, and maintains low basal levels prior to induction. The low basal level of the single copy artificial chromosome, pBeloBAC11, allows for expression of toxic proteins and reduces the possibility of mutation.
A recombinant artificial chromosome, BAC-vvt, encoding the second portion of the Cascade TM system and the vvt gene was constructed and transformed into E.coli TAP expression strain. The result from DNA relaxation assay indicated that the recombinant proteins displayed topoisomerase activity. However, the result from the SDS-PAGE suggested that the toxic proteins were not overexpressed. To tackle this problem, the T7 expression system, E.coli BL21 carrying pET-vvt infected with bacteriophageλCE6, should be used to overexpress the toxic proteins.
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