The introduction of new genetic information into microbial production organisms is one of the keystones of modern biotechnology, this can be achieved i.a. by the introduction of plasmids. Extrachromosomal plasmids replicate autonomously and show a plasmid-specific plasmid copy number (PCN) per cell. In growing cells, a fine-tuned copy number regulation is achieved by a tightly controlled balance of the initiator of replication and the repressor. In theory, the plasmid copy number can be influenced at will by a targeted manipulation of this balance. In order to allow transient PCN changes in growing cells, an inducible, precisely adjustable strategy is needed. In this thesis, cop, the repressor of plasmid replication of pE194, was over-expressed independently from the native regulatory system. The developed overexpression system was used to force a severe imbalance in favor of the repressor, therefore intentionally driving the loss of replicating plasmid from the population. As a consequence, cells that previously incorporated the plasmid into their genome gain a selective advantage as they stably inherit an antibiotic resistance encoded on the now non-replicative plasmid. In established genomic modification strategies, a forced imbalance is dependent on elevated temperatures. However, the established protocols for genomic modifications are time-consuming and require extensive screening for clones with the desired genotype. The aim of this thesis was to conceive and establish a novel vector for a reliable, faster and more efficient protocol with a minimal screening requirement. The promotor controlling the repF-cop operon was characterized by supplying the repressor Cop in trans, revealing a complete repression at high Cop concentrations. Furthermore, the gradual dose-dependency of this repression could be shown. Over several intermediate steps, the novel All-in-one vector pBAio was designed. With this vector, the PCN in growing cells can be controlled and even fine-tuned. Forcing a complete stop of plasmid replication, resulted in a 100% plasmid integration rate that eliminated the need for integrand screening and increased the efficiency (i.e. mutant to wild type ratio). The new, pBAio-based protocol allows for fast and efficient markerless genomic modification in different, industrially relevant Bacillus sp..
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:76957 |
| Date | 13 December 2021 |
| Creators | Stetter, Karen |
| Contributors | Mascher, Thorsten, Schweder, Thomas, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
Page generated in 0.0042 seconds