Overview
In our project, we have devised a novel kill switch based on epigenetic modifications. Our endeavor is focused on ensuring safety measures for engineered microorganisms in a broader range of application environments.
To construct the kill switch, we have designed and built a novel regulatory system based on epigenetic modifications. These parts are made available for utilization by future iGEM teams.
We have established a mathematical model that describes the operational principles of our kill switch within cells. This model will help other iGEM teams in gaining a better understanding of our project.
We have designed a card game aimed at disseminating knowledge of synthetic biology-related technologies and iGEM to the general public.
Additionally, some of the troubles we encountered and resolved during the experimental process have been documented on this page, with the hope that it will be helpful for future iGEM teams.
Figure 1. Overview of our contribution
Safety
With the advancement of synthetic biology, engineered microorganisms hold the potential for application in an increasingly diverse array of scenarios, including food, natural water bodies, and within the human body. Our Wind-up Cell aims to serve as a versatile tool to facilitate the safe deployment of engineered microorganisms in these laboratory-extraneous contexts. In this project, we have developed a whole-cell test paper as an illustrative example of the practical application of the Wind-up Cell. Additionally, we hope that our kill switch can contribute to enhancing the safety of future iGEM teams' projects.
For more detailed information about our contributions in biosafety, please refer to the Safety Page.
Figure 2. Our Wind-up Cell aims to serve as a versatile tool to facilitate the safe deployment of engineered microorganisms in these laboratory-extraneous contexts.
New Parts
In Saccharomyces cerevisiae, leveraging the target specificity of dCas9 and the histone deacetylase activity of Sir2, we designed and built a dCas9-Sir2 fusion protein(BBa_K4703016). In Escherichia coli, we have constructed a fusion protein comprising dCas9 and Dam methylase, controlled by the trc promoter(BBa_K4703002).These two fusion proteins possess both the sgRNA targeting function of dCas9 and the epigenetic modification capability of Dam methylase, which can potentially assist future iGEM teams aiming to achieve site-specific epigenetic modifications at a target loci.
Figure 3. dCas9-Sir2 fusion protein expression cassette
Figure 4. dCas9-Dam fusion protein expression cassette
Model
Simultaneously, we have developed a macroscopic Cell Death Simulation Model to elucidate the process of cell death initiation upon removal of the inducer in contact with an external solution.
In our project, we have established a microscale toxic gene expression model to elucidate the induction process of toxic genes' expression pathways.In this model, we have configured controls for each parameter with adjustable values (see Software Page for details). This enables convenient parameter adjustment for predictions when replacing different strength promoters or epigenetic modification tools during wet lab experiments. Furthermore, other iGEM teams utilizing epigenetic modifications for gene expression regulation can effortlessly utilize our model for predictions.
More details about our models are documented on Model Page.
Figure 5. Controls of toxic gene expression model
CRISPR-Related Card Game
We have designed a board game promoting biosafety awareness and related to the principles of CRISPR and epigenetic modifications titled "Engineered Microorganism: Under the Sword of Damocles." This game draws inspiration from the turn-based gameplay of Werewolf, but all its characters are intricately related to biology and adhere strictly to biological principles. Through this game, we aim to provide players with a deeper understanding of the fundamental knowledge of CRISPR/Cas9, while simultaneously igniting their passion for synthetic biology.
During gameplay, players will assume roles from three unique factions: the Gene Faction, Cutting Faction, and Neutral Faction. Each faction's characters have their distinct missions. Players must skillfully disguise their own identities and discern others' deceptions to eliminate players from opposing factions. Notably, each game card is imprinted with synthetic biology knowledge related to its character, allowing players to learn while being entertained.
The board game's card design, rules, and other details are extensively documented on the Education Page. We also warmly welcome other iGEM teams to use our board game for the dissemination of biological knowledge or to build upon it, enriching the content of the game.
Figure 6. The cards of Engineered Microorganism: Under the Sword of Damocles.
Troubleshooting
1. When constructing the plasmid pR_DK09 (BBa_K4703036), we attempted linearization of the plasmid BBa_K4703035 via PCR. However, the electrophoresis results of the PCR product displayed numerous non-specific amplification bands, and modifications to the PCR reaction conditions and program did not significantly improve the situation. Subsequently, this issue was resolved by changing the primers (Table 1).
Table 1. We made modifications to the primers.
Furthermore, when constructing the dCas-Dam fusion protein (BBa_K4703002) using pR_DK09 as the vector, we encountered non-specific amplification in the gel electrophoresis of vector PCR product. This issue was resolved by using BamHI enzyme cleavage instead. Therefore, we do not recommend PCR-based linearization for other iGEM teams looking to modify BBa_K4703036 or BBa_K4703035.
2.When conducting practical simulations using our Cell Death Simulation Model, due to limitations in our computer's processing power, we encountered challenges in performing computations for the complete model. To mitigate this issue, made several approximations and assumptions, which are documented on our Model Page. We hope that these insights can serve as inspiration for other iGEM teams facing similar computational constraints.
