During our time in the lab we mainly focused on proving the two most important aspects of our biocontainment system in order to thereby show our proof-of-concept. This firstly includes the possibility of utilizing two independently replicating plasmids in one bacterium. Secondly, we evaluated the toxicity of the selected toxins to the bacteria. We consider the potency to kill and the stability of the system to be the most important criteria for a successful biocontainment system. By proving these pivotal building blocks of DOPL LOCK are effective, we can ensure a reliable foundation with high stability and the capacity to kill the bacteria for our final biocontainment system. This essential foundation proves the build-up of DOPL LOCK, as we designed it, is feasible with additional time.
With the two main criteria for the proof-of-concept now fulfilled, we designed the final stage of the proof-of-concept for DOPL LOCK. Unfortunately, we were not able to finalize the assembly of the entire system due to time constraints. However, with the acquired results, we propose experiments and further engineering to develop the full DOPL LOCK system and assess its effectiveness.
To develop our proof-of-concept further into the fully designed DOPL LOCK system, we have come up with a variety of experiments. First, to prove that the plasmid transfer of one of the plasmids leads to cell death and to assay the genetic stability of DOPL LOCK, we have proposed a design of these plasmids. As shown in Figure 3, the first plasmid contains the CcdB toxin and SOK antitoxin in the pJUMP29 backbone, with the pBR322/ROB Ori. The second plasmid contains the HOK toxin and the CcdA antitoxin in the pJUMP46 backbone, with the p15A Ori. Lastly, we've thought about experiments to further investigate the physical containment aspect of the DOPL LOCK system. We believe that these experiments will help turn our current proof-of-concept into a fully realised biocontainment system.
Figure 3: A schematic representation of the design for testing the proof-of-concept. The plasmid containing the pBR322/ROB Ori is expressing the CcdB toxin under the control of the promoter J23104 and the SOK antitoxin under the control of the promoter J23100. The plasmid containing the p15A Ori is expressing the HOK toxin under the control of the promoter J23104 and the CcdA antitoxin under the control of the promoter J23100.
To be able to make the most impact with DOPL LOCK, we want it to be easy to implement and widely applicable. Therefore, our goal is to add DOPL LOCK as a part to the SEVA plasmid nomenclature. Our biocontainment modules will be added to the SEVA nomenclature as a potential substitute for the region of the plasmid containing the origin of transfer (OriT) and the antibiotic resistance gene. The OriT allows for conjugational mobilization of pSEVA plasmids into organisms for which no alternative transformation procedures are available . Conjugation is one of the most well understood mechanisms of HGT . By removing the OriT we could reduce the risk of HGT to protect the integrity of an ecosystem. With DOPL LOCK we want to eliminate this risk by eliminating the OriT and replacing this with our DOPL LOCK biocontainment module (Figure 7).
Additionally, we want to replace the antibiotic resistance cassette of the plasmids, with a split GFP as a selection marker of the presence of both plasmids (Figure 7). By using a split GFP selection marker the DNA coding for the subunit 11 of GFP is present on the plasmid harboring the gene of interest, while the other plasmid is harboring the DNA for the first 10 subunits of GFP. When both parts of the protein are expressed, GFP signals can be observed .
Figure 7: Proposed incorporation of DOPL LOCK in the SEVA nomenclature. Where the OriT is replaced with a biocontainment module and the antibiotic selection cassette can be replaced with a split GFP marker. This plasmid can be co-transformed with the plasmid harboring the split GFP and biocontainment module counter parts (not shown) to apply the biocontainment strategy.
With this proposed implementation, we want to provide a transparent and open-source platform that could be used for the application of SEVA plasmid-based GMOs in a semi-contained environment. These semi-controlled applications of GMOs entails applications in an environment where localized containment is possible, yet with a clear physical path for GMOs to spread into the environment, for example wastewater treatment plants, nitrogen fixation in plants or bioremediation of contaminated soil as described in our Implementation page.
- Silva-Rocha, R., Martínez-García, E., Calles, B., Chavarría, M., Arce-Rodríguez, A., de Las Heras, A., Páez-Espino, A. D., Durante-Rodríguez, G., Kim, J., Nikel, P. I., Platero, R., & de Lorenzo, V. (2013). The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic acids research, 41(Database issue), D666–D675. https://doi.org/10.1093/nar/gks1119"
- Muteeb, G., & Sen, R. (2010). Random mutagenesis using a mutator strain. Methods in molecular biology (Clifton, N.J.), 634, 411–419. https://doi.org/10.1007/978-1-60761-652-8_29
- Drake, J. W., Charlesworth, B., Charlesworth, D., & Crow, J. F. (1998). Rates of spontaneous mutation. Genetics, 148(4), 1667–1686. https://doi.org/10.1093/genetics/148.4.1667
- Nguyen, H. H., Park, J., Park, S. J., Lee, C. S., Hwang, S., Shin, Y. B., Ha, T. H., & Kim, M. (2018). Long-Term Stability and Integrity of Plasmid-Based DNA Data Storage. Polymers, 10(1), 28. https://doi.org/10.3390/polym10010028
- Miller JH. A Short Course in Bacterial Genetics. Harbor, NY: Cold Spring; 1992
- Burmeister A. R. (2015). Horizontal Gene Transfer. Evolution, medicine, and public health, 2015(1), 193–194. https://doi.org/10.1093/emph/eov018
- Ceccarelli, D., Daccord, A., René, M., & Burrus, V. (2008). Identification of the origin of transfer (oriT) and a new gene required for mobilization of the SXT/R391 family of integrating conjugative elements. Journal of bacteriology, 190(15), 5328–5338. https://doi.org/10.1128/JB.00150-08
- Romei, M. G., & Boxer, S. G. (2019). Split Green Fluorescent Proteins: Scope, Limitations, and Outlook. Annual review of biophysics, 48, 19–44. https://doi.org/10.1146/annurev-biophys-051013-022846