Team:TAU Israel/Future Plans Biology
Future Plans - Biology
Our Vision
From the beginning of our work, we were well aware of our ambitious goal, and there are many more things we wish we could have accomplished in the timespan of the competition. While we have successfully reached most of our goals for the competition, we like to view this project as the beginning of a much more significant innovation.
As such, we would like to highlight the most prominent future directions that are currently in progress.
Full plasmid generator
Currently, our software generates a full expression cassette which includes both an optimized open reading frame and a promoter according to user-defined preferences.
However, this genetic element is not transformable on it’s own - there are a few more components that still need to be incorporated into it, such as origin-of-replication optimization (which is currently analysed and modeled for the next version of our software). The ability to optimize an entire plasmid using our tool would be a significant leap in our model’s capabilities, and we consider it to be the top priority in any future work.
Kill-switch
While our solution significantly increases the level of safety in the introduction of engineered bacteria into a community, additional safety measures must be taken in order to ensure with absolute certainty that no engineered bacteria will spread beyond its predefined environment. Additionally, the ability to disable genetic circuits on command may also be desirable in an industrial environment, where controlling the lifespan of engineered bacteria provided to customers can be a useful tool.
A kill-switch is a genetic circuit that can essentially act as an “emergency button” - selectively eliminating the engineered bacteria (or engineered machinery within said bacteria) on command, preventing it from spreading any further, while also enabling precise spatial and temporal control over the presence of the engineered bacteria in an environment.
The topic of kill-switches was already addressed by previous iGEM teams, each coming up with very different solutions. Systems such as the mazEF toxin-antitoxin system proposed by the 2020 TU Darmstadt team and the self-digesting plasmid system designed by the 2012 Paris Bettencourt team are quite compatible with our project, and we are looking into building upon their previous work and incorporating such a mechanism into our service in the future.
Transformation- SEGA technology
While recombinant plasmids may be the most widespread method of handling DNA in bacteria today, chromosomal gene expression methods are becoming increasingly more commonplace in some fields, as the advancement of research exposes many of the inherent disadvantages of recombinant plasmids [1], as the increased stability [2] and lower metabolic burden [3] offered by genome integration becomes more relevant. Recently, a paper published in Nature Communications has outlined an innovative, yet simple methodology of bacterial genome engineering that is highly standardized and very broadly applicable named the Standardized Genome Architecture (SEGA) [4]. We believe that integration with such emerging transformation platforms would help us cross the transformation barrier and apply our technology in various environments.
Biology
Fusion PCR
The OIL-PCR method by Peter J. Diebold et al. [8] allows us to assess the abundance of bacteria that contain the plasmid-encoded GOI in a microbial community, which potentially reveals if our approach is able to restrict GOI abundance governed by HGT events.
We managed to begin this method towards the end of the iGEM season, and the results of the calibration can be found on our Results page. In the future, we would like to continue by cleaning the fused product (GOI-16S rRNA) and amplify it with a specific set of forward primers targeting the fusion regions and species-specific reverse primer, targeting a variable region of the 16s rRNA gene via qPCR to identify the bacterial species.
Additionally, we are planning to expand this method to combine GOI abundance and its expression by applying the same principles of GOI-abundance method but based on fusion-PCR of mRNA products of GOI and 16s rRNA genes. For this purpose, the fusion-PCR step will include primers targeting GOI’s mRNA, with a reverse primer having a tail complementary to the mRNA of 16s rRNA and reverse transcriptase to establish the fused product. Potentially, this method can detect both the host identity and its GOI expression levels.
Since the analysis and testing we conducted during this competition showed very promising results, we believe that further analysis of the model should be conducted to further prove its efficacy, conducting broader assays and tests with more species of bacteria (such as species that are more closely related genetically), testing with a wider variety of genes of interest, a wider variety of promoters, etc. and we are very positive about the success of such future efforts. Additionally, further analysis and testing of different scoring algorithms in the model itself may also prove to be beneficial.
Another aspect that we have set in our sights as a future target is finding out the relation between the different model components - how do changes in multiple model components (i.e., a change in both the ORF and promoters) affect the final result when applied simultaneously? This is a topic that was not part of the scope of our analysis for this competition, but considering our success during the competition we have set it as a goal for our next step.
Synthetic Microbiomes
While our project discusses the engineering of particular bacteria in an existing community, current research is also being conducted on the applications of entirely synthetic microbial communities [5-7]. We believe that our tool can be of great use when trying to engineer such synthetic communities, as it greatly improves the ability to engineer particular bacteria species in the community into different roles, improving the efficiency and stability of the community. As such, we consider synthetic microbiomes to be one of the most promising applications of our project in the future, and further work in this field is in our sights.
Practical Applications
Besides the scientific applications of our project, we have begun looking for suitable practical applications for our project. Through our human practices work, we have already outlined a few possible real-life use cases, and we are very excited to start working on some of those applications in the future.
References
- B. Shao, J. Rammohan, D. A. Anderson, N. Alperovich, D. Ross, and C. A. Voigt, “Single-cell measurement of plasmid copy number and promoter activity,” Nature Communications, vol. 12, no. 1, 2021.
- B. Ou, C. Garcia, Y. Wang, W. Zhang, and G. Zhu, “Techniques for chromosomal integration and expression optimization in Escherichia coli,” Biotechnology and Bioengineering, vol. 115, no. 10, pp. 2467–2478, 2018.
- S. Birnbaum and J. E. Bailey, “Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli,” Biotechnology and Bioengineering, vol. 37, no. 8, pp. 736–745, 1991.
- C. N. Bayer, M. Rennig, A. K. Ehrmann, and M. H. Nørholm, “A standardized genome architecture for bacterial synthetic biology (SEGA),” Nature Communications, vol. 12, no. 1, 2021.
- R. L. Clark, B. M. Connors, D. M. Stevenson, S. E. Hromada, J. J. Hamilton, D. Amador-Noguez, and O. S. Venturelli, “Design of synthetic human gut microbiome assembly and butyrate production,” Nature Communications, vol. 12, no. 1, 2021.
- R. S. de Souza, J. S. Armanhi, and P. Arruda, “From microbiome to traits: Designing synthetic microbial communities for improved crop resiliency,” Frontiers in Plant Science, vol. 11, 2020.
- J. Ke, B. Wang, and Y. Yoshikuni, “Microbiome Engineering: Synthetic Biology of plant-associated microbiomes in Sustainable Agriculture,” Trends in Biotechnology, vol. 39, no. 3, pp. 244–261, 2021.
- P. J. Diebold, F. N. New, M. Hovan, M. J. Satlin, and I. L. Brito, “Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR,” eLife, vol. 10, 2021.
