Strategies to control plasmid copy number
Silvia Löbsch
2010
unpublished
Gene therapy holds its promises to silence dysfunctional genes on either transcriptional or translational level or by replacement with functional genes. Not long ago it has revolutionized modern medicine and since then thousand clinical trials have been authorized worldwide. To put the medical profit of gene therapy and DNA vaccination into practice, methods to produce highly pure plasmid DNA (pDNA) free from bacterial chromosomal DNA, RNA, proteins and endotoxins have to be developed. Since it
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... is known that plasmid copy number (PCN) and plasmid stability are affecting the success of gene therapy to a large extent, the rational design of an optimized vector is an ultimate ambition. To prevent excessive plasmid replication, posing metabolic burden resulting in growth inhibition right up to cell death to the workhorse Escherichia coli (E. coli), regulation of PCN is to be considered in the rational design of any vector as well. In this work the main goal was to elaborate a system that regulates plasmid replication of ColE1-type plasmids in Escherichia Coli (E. coli) by overexpressing tRNAAlaU and rssB encoding genes. In the first approach the wt and mutated tRNAAlaU gene, whose expression was controlled by the T7 promoter, was inserted into the E. coli chromosome. In shake flask experiments overexpression of the tRNAAlaU gene was induced by addition of IPTG and subsequently its effect on three different ColE1-type plasmids has been tested. In the second approach, overexpression of rssB, whose expression was also controlled by the T7 promoter, should result in enhanced plasmid concentration after IPTG induction whereas the biological mechanism remains unclear. In shake flask experiments, rssB overexpression was also induced by adding IPTG. All in all, the experimentally gained data have demonstrated clearly that overexpression of rssB and tRNAAlaU encoding genes resulted in enhanced plasmid concentration of ColE1-type plasmids.
doi:10.25365/thesis.12378
fatcat:657zjdyycnagzkpspeqrr222fu