title: Establishing the fast-growing bacterium Vibrio natriegens as a next-generation chassis for synthetic biology
Creators: name: Stukenberg, Daniel
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: Life sciences
Biowissenschaften, Biologie

description: Vibrio natriegens is the fastest-growing organism known to date with a minimal doubling time of less than ten minutes under optimal conditions. Due to this exciting property and also because of its capability to use a wide range of carbon sources, it was proposed as a novel chassis for Synthetic Biology and Biotechnology. However, so far reports about successful applications of V. natriegens are scarce. This is likely due to the lack of efficient and reliable genetic tools. Furthermore, the knowledge about the biology of V. natriegens is still limited, which leads to unforeseeable challenges for its application. In combination, these two reasons have prevented the widespread application of V. natriegens in Synthetic Biology so far.
In this cumulative thesis I describe my contribution to tackling these limitations. 
Within the scope of this thesis, two CRISPR-Cas9-based tools were added to the set of available methods for V. natriegens. 
The first tool is NT-CRISPR. This method was developed to improve our ability to perform genomic modifications in V. natriegens. NT-CRISPR relies on V. natriegens’ ability to take up free DNA from the environment in a process called natural transformation. This can be used to introduce a wide range of genomic modifications, such as deletions, integration of foreign DNA or the introduction of point mutations. In a subsequent step, CRISPR-Cas9 is activated to selectively kill non-modified cells, thereby drastically increasing the efficiency of genome modification by natural transformation. It was demonstrated that genome engineering can be performed with high efficiencies for various types of modifications, as well as for the simultaneous deletion of three sequences.
In addition to NT-CRISPR, which leads to permanent genetic modifications, graded-CRISPRi was developed as a tool for the inducible repression of genes in V. natriegens. By using two different inducible promoters, a tightly regulated system was created, which does not show any activity in the absence of the inducers. Furthermore, libraries with gRNAs of different lengths and mismatches to the target sequence were used to generate graded knockdown strengths. This concept was applied to four different reporter genes to demonstrate that graded-CRISPRi can be used to generate various expression levels of multiple targets in V. natriegens. Lastly, graded-CRISPRi was targeted against native genes to study the relationship between protein abundance and growth behavior.
In addition to developing genetic tools, some progress has been made regarding the understanding of the rapid growth of V. natriegens. Like almost all strains of the Vibrionaceae family, V. natriegens has a bipartite genome configuration. The two chromosomes were fused to generate the monopartite strain synSC1.0. A phenotypic characterization in comparison to the parental strain revealed that synSC1.0 did not show any major difference. This led to the conclusion that the bipartite genome configuration of V. natriegens is not a requirement for its rapid growth.