Where do we go from here?
During our project, we were using the Vmax express strain of V. natriegens. We discovered too late in our project, from Rene Inckemann and Daneil Stuckenburg, that the WT strain of V. natriegens is much easier to work with. Future work would be undertaken using the WT strain with appropriate knockouts to enable transformation.
We plan to further optimise our media in the future by testing a broader scope of ingredients. We have reason to believe \(\ce{Na2HPO4}\) and brain-heart infusion are beneficial ingredients, as they have been seen in media that are faster than ours. For future optimisation experiments, we plan to use optimal levels of ingredients we have already tested, and test a broader range of ingredients with them.
Unfortunately, long delivery times meant we started cloning much later in the summer than we had hoped. Because of this, future work should firstly aim to test the activities of our three plasmids – the MutaT7 test cassette and sorbitol rescue test to validate our mutagenesis and selection approaches. We would also aim to characterise our collection of growth slowing genes, to see if negative selection with the use of a riboswitch would be feasible. We would probably swap out our Lac promoters for a more easy to induce promoter as this caused many obstacles when carrying out these validation assays. After confirming all our components work separately, we would test rEvolver plasmids together to see if the whole system is capable of working in a single cell.
We hope to carry on rEvolver into next year's iteration of iGEM and as our project developed, we came up with many changes we would like to make to our final plasmid design.
Firstly, the MutaT7 RNA polymerase base editor fusion plasmid was very large. We would consider in the future using ribozyme splicing to allow each fusion so we would only have to incorporate the large T7 RNA polymerase once, whilst still producing both base editor fusions. Secondly, we realised transforming cells with three plasmids may cause issues. To work around this, we will use NT-CRISPR/Cas9 to knock out base-excision repair pathways permanently abolishing the need for the CRISPRi plasmid, which would have to remain in the cell to continuously knockdown these repair pathways. NT-CRISPR/Cas9 works by using natural transformation (NT) to knockout the genes. The gRNAs then target Cas9 to WT genes which will kill unedited cells. This provides a means to counterselect for cells that have not undergone editing, creating a populating deficient in repair pathways to increase the efficiency of mutagenesis – reducing our cumbersome three plasmid system to a two plasmid system.
Our MutaT7 has shown the ability to directly evolve protein coding genes. However, since MutaT7 mutagenesis is directly coupled to transcription of genes, the evolution here is limited to open reading frames – therefore unable to perform evolution of promoters, terminators and other important regulatory elements. Therefore, an alternative method must be used for the improvement of these constructs. HiSCRIBE is a new retrotransposon based gene editing system, capable of evolving regulatory elements. Currently, the MutaT7 system is only capable of evolving protein coding regions to which the RNA polymerase can bind so adopting this technology for rEvolver will broaden our scope for directed evolution. HiSCRIBE is yet to be used for directed evolution, so this is something we are very excited to start implementing.
Though the bioreactor has already reached a state in which it can culture aerobic, heterotrophic organisms, expanding its capabilities to culture anaerobes, eukaryotes, and photosynthetic organisms could greatly increase its applicability. These adaptations may require new materials (perhaps ones that allow an airtight seal to be made) and improved control of parameters like agitation rate and gas-flow. To culture photosynthetic organisms, a regulated light-source could be added, but this would require new power and control electronics. Depending on the final dimensions of the bioreactor, parameters like height, thickness, and light-positioning may need to be adjusted to avoid self-shading.
Thinking in another direction, it could be interesting to scale up our system. One way this could be done is by linking multiple bioreactors into an array using peristaltic pumps. This could allow for different stages of culturing, so cells could be cultured in one media before being syphoned out into another vessel with a different composition (perhaps moving from growth to induction media). This could also allow for the selection of evolutionary candidates via multiple mechanisms — in one reactor after another.
From a practical standpoint, replacing laser cut components and materials with 3D-printed variants could contribute to increased accessibility and reproducibility. The bioreactor subsystems could also have their performance improved in a number of ways. The OD measurement system, for example, could integrate a laser for reduced refractive noise.
Finally, Internet of Things (IoT) integration for remote monitoring and control of the reactor could be achieved by moving to the Wi-Fi equipped W variant of the Raspberry Pi Pico.
Particularly after our human practice chats with Michael Magaraci, it was obvious that there is a dearth of directed evolution tools for eukaryotic cells. This can often cause a problem with the functionality you are trying to evolve is unique to eukaryotes. To keep with our theme of working in fast replicating organisms, we can look into expanding our rEvolver toolkit into Kluyveromyces marxianus — a super fast-growing eukaryote with a 52 minute doubling time (compared to S. cerevisae which takes 90 minutes to double)!
Our bioinformatics toolbox has a wide array of basic functionalities already implemented, but in the future we plan on adding a few common functionalities and a great deal of complex functionalities to address the very specific needs of scientists across many life science disciplines.
Some basic functionalities we plan on implementing in the very near future include:
Some more advanced functionalities that we intend on implementing:
Eventually we plan on implementing some highly complex features, such as:
Stukenberg, D., Hoff, J., Faber, A. et al. NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens. Commun Biol 5, 265 (2022). https://doi.org/10.1038/s42003-022-03150-0
Farzadfard, F., Gharaei, N., Citorik, R.J., and Lu, T.K. (2021). Efficient retroelement-mediated DNA writing in bacteria. Cell Systems 12, 860-872.e5. 10.1016/j.cels.2021.07.001.