Realizing V. natriegens as a widely used host organism for synthetic biology requires well-funded knowledge about it! Realizing this, we prioritized fundamental research early on. We showed the unparalleled speed of V. natriegens replication, defined a range of optimal growth conditions, including pH and salt tolerance, and the ease of its genetic accessibility. protocols
We managed to enable transformation demonstrated for with high electroporation efficiency and heat-shock transformation to drive synthetic biology research.
In combination with our Marburg-Collection, we accomplished cloning of simple plasmids, from transformation to miniprep, under 12 hours, and assembly and preparation of level 2 golden gate constructs in under three days!
Additionally, our team successfully implemented five fragment Gibson cloning as well as Aquand cloning and achieved high reliability at high performances.
We sequenced both chromosomes with Illumina sequencing, mapped them to existing genome maps and ran automated annotation tools to identify genetic features.
Working concentrations for most common antibiotics were elucidated and used throughout the project.
Applying several electron microscopic methods, we could, apart from generating nice pictures, highlight shape, form and volume of V. natriegens. Fortunately, we could observe several cell divisions in mid process.
constitutive and inducible promoters, RBS, reporter and tools for genome engineering, terminators, oris, resistance cassettes and a set of self-designed connectors.
All parts were submitted to the registry to help future iGEM teams in achieving ambitious projects and, for increased convenience, we additionally enable download of plasmid maps of all parts in our wiki
To characterize our parts, we established a fast and convenient platereader workflow tailored to species-specific properties of V. natriegens and evaluated the optimal plasmidal context.
Little characterization has been done for genetic parts in V. natriegens, so we applied our own workflow to obtain the very first experimental data for promoter strength, dose dependency of inducible promoters, RBS strength, terminator read-through and the insulating behavior of our novel connector parts.
We characterized our ori parts in V. natriegens by showing their impact on reporter expression and furthermore, qPCR experiments revealed differences in plasmid copy numbers depending on reporter expression.
To additionally ease Golden-Gate cloning we developed the software tool Click ‘n’ Clone which provides a GUI in which a user can simply select the desired parts for building a plasmid. A detailed pipetting protocol for manual operation or a picking list that is compatible with lab automation is given as result.
For VibriClone 1.0, we could successfully delete the nuclease dns and create linear DNA cassettes for further genomic modifications, to improve this strain and allow a highly efficient cloning. Moreover we designed VibriClone 2.0 with additional features, such as deletion of both nucleases, a recA1 mutation, cold resistance and the ability for blue white selection, which makes our strain more suitable as the next generation cloning chassis.
In case of VibriXpress we detected a protease with a high similarity to the Lon protease of E. coli that needs to be deleted for high protein yield and could design and create cassettes for the integration of the T7 polymerase, the deletion of lon and the deletion of dns. We were not able to cotransform these fragments into V. natriegens, but could show, that the integration of the dns deletion cassette was successful.
We created the strain VibriInteract carrying the deletion of cyaA. The excision of the selection marker, using the Flp/frt system and curing the plasmid for natural transformation was successful. By this, we provided a strain for fast protein interaction studies, could demonstrate its functionality by V2H assays and characteized its growth and cell morphology.
Additionally we detected and characterized 15 possible sites for genomic integrations.