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.
We designed and constructed the Marburg Collection the most flexible golden-gate based toolbox for prokaryotes. It contains 123 LVL0 parts including:
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 from 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.
Few 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 readthrough 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 and. A detailed pipetting protocol for manual operation or a picking list that is compatible with lab automation is given as result.