The production of topoisomerase I starts with pTrc99A and λZAPII/topAcysB, the templates of instruction for the enzymes molecular makeup. Incorporating the gene of interest into the vector requires the proper ligation that utilizes restriction sites to cut specific sites allowing the integration of topAcysB gene into an operable pTrc99A. The new plasmid enters a bacterial host through transformation in order to achieve the expression of the topA gene. Selection pressures are used to obtain the proper recombinant pTrc99A/topAcysB plasmid within a cell. Transformant cell cultures induced with IPTG, stimulate the production of topoisomerase I. Once IPTG binds to the lac repressor, it makes way for the trc promoter and topoisomerase I is produced. Topoisomerase I is extracted out the cell and compared to uninduced cells to verify the mechanisms that read and express the topA gene within pTrc99A. Lowry assay, polymerase chain reaction, absorption assays, and gel electrophoresis are implemented to view the fragments found in recombinant cells that encode topA gene and produce active topoisomerase I.
Topoisomerase I is an enzyme that uses its unique structure to relax supercoiled DNA in prokaryotic and eukaryotic cells. Typically, topoisomerase I removes negative coils by units of one, and is often found at sites that are undergoing replication or transcription. Topoisomerase I is made up of four domains that contribute to the enzymatic activity. The active site in topoisomerase I binds to the nucleotides near the Rossmann fold in domain I, cutting the DNA strand, and relaxing the DNA. Although there are 865 amino acids that make up topoisomerase I, there are s...
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...thin pTrc99A/topAcysB plasmid, then the primers would not adhere to the exclusive sites found within topAcysB gene and the ligation of this product into M13mpI9 would not happen. To verify whether topAcysB was cloned, the multiple cloning site embedded in the lacZ ' gene of M13mp19 prevents alpha-complementation when DNA is cloned into one of the restriction sites.(1) Therefore, a blue plaque on media forms when M13mpI9/topAcysB is not ligated successfully, and white plaques will appear if M13mpI9/topAcysB was ligated successfully. The plates retrieved from this procedure exhibited white plaques, data not shown in the results, but having white plaques present, illustrates that topA gene had been cloned through PCR. The pTrc99A/topAcysB plasmid has been proven to be a useful expression vector, and has multiple applications in the process of forming topoisomerase I.
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