Overview
This year's AIS-China project aims to provide a novel solution for the prevention and control of mosquito-borne diseases, with the project initiation goal of offering an additional solution for environmental safety and human health. Throughout the process of advancing the project, we value feedback from stakeholders and integrate "safety" into every aspect of our project.
Firstly, in the use of live E. coli, we have engineered a "kill switch" to address potential end-users' concerns, laying the foundation for the safe application of our Genetically Modified Organisms (GMOs). And our testing results indicate the X174E inducible expression system is recommended.
Based on this, we have established a Controllable Automatic Droplet dispenser Moduleto facilitate the entry of GMO-containing devices into the real world.
Of course, safety is paramount. In the engineering, testing, and construction of hardware, under the guidance of experts, we have assessed the risks and provided corresponding training to our team members, demonstrating our team's commitment to laboratory safety.
By integrating the X174E inducible expression system with the Droplet dispenser Module, we have created a comprehensive safety system for the external use of GMOs that can be utilized by any future iGEM team or research group. We will provide a more detailed description of this in the following sections.
Figure 1. Integrated safety system built by 2024 AIS-China Team
X174E inducible expression system in E. coli
To attract blood-seeking mosquitoes, we intend to deploy HMBPP, a Plasmodium-derived metabolite, produced by live, genetically modified E. coli, and inactivated yeast cells expressing shRNA to eliminate the insects. However, feedback from public surveys and consultations with Shenzhen CDC and Chinese environmental ministry officials revealed lingering concerns about genetically modified organisms.
Committed to environmental safety, we engineered a hardware-based containment system equipped with kill switch for live E. coli. After extensive research of kill switch mechanisms, autolysis genes are chosen and engineered to be IPTG-inducibly expressed and tested, including T4L(BBa_K5186018), Pa-T4L(BBa_K5186019), 2Pa-T4L(BBa_K5186020), X174E(BBa_K1835500). Finally, our tests show that X174E outperforms with the majority of cell lysis within one hour of induction, which makes it an optimal candidate for our kill switch component.
This X174E inducible expression system, recommended by the 2024 AIS-China team, can be utilized by other iGEM teams and researchers who need to establish a kill switch for the controlled restriction of GMOs in their projects.
Figure 2. Various autolytic genes expression cassettes are engineered for kill switch setting in E. coli strain DH5a. (a) Genetic circuit construction of strains KS 1-4. (b) Gel electrophoresis analysis of transformed autolytic genes expression cassettes. (c) Growth curve of control (E. coli strain DH5a) and strains KS 1-4.
Droplet dispenser module in device
To ensure the feasibility of our project and functionality of our product, we carefully examined feedback received from stakeholders. In feedback, we noticed that our stakeholders want us to ensure biosafety and minimize the risk of leakage of bacteria used in our hardware. With this consideration, we designed the hardware to fully enclose the living bacteria, and added a controllable automatic drip module to the device.
We've already opened sourced our Droplet dispenser module assembly and implementation methods. For details, check our Hardware page. By adopting our kill switch mechanism, future iGEM teams and stakeholders can ensure safer and more responsible microbial management, promoting environmental safety.
Figure 3. Integration of Screw Motor, Syringe, and Serum tube
Lab safety
Although we have dived into ways to fully enclose living bacteria in our device, committed to biosafety, AIS-China emphasizes the importance of maintaining a safe laboratory environment. We achieve this by providing regular biosafety training to our team members, thereby mitigating potential risks proactively.
For testing the effectiveness of HMBPP and our shRNAs, we completed the check-in form and applied to conduct mosquitoes related experiments. To better cope with mosquitoes in the lab, we engaged with Professor Xiaoguang Chen's team at Southern Medical University. Following their advice, we made a training handbook and safety management booklet for experiments involving mosquitoes, using them to conduct training within our team.
Management of BSL1
Our laboratory operates under BSL 1 conditions, primarily handling E. coli and S. cerevisiae. We are well-equipped with essential personal protective equipment (PPE), including lab coats, masks, gloves, and safety goggles. Stringent segregation is enforced, particularly between resting areas and laboratory spaces, with extra caution around electrophoresis areas due to the potential hazards of gel dye coming into direct human contact.
We have implemented comprehensive safety training sessions to equip our team with emergency response protocols and the safe operation of various lab equipment. For equipment posing a risk of injury, we have affixed instructions and precautionary tips to mitigate potential accidents.
Additionally, we have established a rigorous waste management system to prevent contamination and infection. Specifically, for disposable and reusable containers from bacterial cultures, we follow a systematic treatment process that involves disinfection and segregation for appropriate disposal or cleaning and reuse.
Management of Mosquitoes
Initially, we engaged in online collaboration with Professor Chen Xiaoguang's team, specializing in mosquito management. Despite this, we faced challenges in mosquito breeding, leading to the loss of two experimental batches.
Eventually, we secured an offline communication, where we observed their meticulous approaches. Their comprehensive mosquito management system was impressive.
Drawing from their expertise, we refined our laboratory training protocols for mosquito breeding experiments. Additionally, we compiled instructional slides to assist future teams or researches facing similar challenges during their iGEM journey.