SAFETY AND SECURITY
General Safety
Our laboratory work is conducted at Lab 106 (Synthetic Biology and Biofabrication Laboratory) of National Taiwan University’s department of Biochemical Engineering. The lab is a Biosafety Level 1 lab operated by Professor Hsuan-Chen Wu [1], that only permits the use of agents that don’t pose significant health risks upon exposure. Our safety were conducted under the supervision of graduate-level students and trained lab technicians. By only working with the common bacterial E. coli strain MG1655 and strain DH5α, we eliminated any or most general biosafety and health concerns that would otherwise arise if other bacteria strains were chosen.
Before performing lab work, we made experiment plans that were reviewed and validated by our instructors and advisors to prevent unnecessary risks. In addition to safe and organized laboratory practices, we also completed four hours of online training provided by the Taiwanese government’s Taiwan Centers for Disease Control [2]. This training covered topics of laboratory safety, risk groups, risk assessment, biosafety program management, biosecurity, and more. All plans are uploaded onto a shared drive and documented in a loose leaf binder kept in the lab to keep instructors and team members informed of the experiment details and schedules. We made certain that all general lab-safety guidelines of Lab 106 were followed and all equipment was used with caution. safety involving microorganisms were conducted in laminar flow hoods, and the glassware used was disinfected with diluted bleach and rubbing alcohol upon experiment completion. In the case of bacterial contact with our workspace, rubbing alcohol was immediately applied to any contaminated areas and wiped clean. Chemical waste was disposed of in biohazard waste bins; chemicals, pipette tips, and glassware were sterilized in the autoclave.
Project Safety
We recognize the potential hazards of introducing genetically engineered organisms to the equilibrium of the coral reef microenvironment.
The key potential hazards that need to be addressed are:
Figure 5. Potential hazards caused by genetically engineered bacteria
Our project, through extensive research and expert consultation, has tailored specific designs to address these potential biohazards:
1. Disruption to the Holobiont
After extensive consultations with esteemed experts in the field, including Dr. Tang Sen-Lin [3] of the Marine Microbial Ecology Lab under the Academia Sinica Biodiversity Research Center, Professor David Bourne [4] of the James Cook University Marine and Aquaculture Sciences department, and Dr. Lone Hoj [5], a coral probiotics specialist of the Australian Institute of Marine Science, we made the choice to utilize the following two designs for their non-disruptive attributes:
  1. E. coli Biofilm: As E. coli is one of the most well-studied microorganisms and easily propagated, we’ve utilized E. coli in our design for surface colonization. Our choice of using biofilm produced by E. coli ensures that our delivery mechanism is safe and sustainable to the environment because of its biodegradable and non-toxic nature as well as its properties being well characterized.
  2. SAR11: Constituting approximately a quarter of oceanic prokaryotic life, we selected SAR11 as our biofilm-producing bacteria specifically for its prevalence.
  3. The SAR11 bacteria not only plays a significant role in the coral diet, but ensures the safe assimilation of our product into the coral holobiont—all while remaining free of any disruptive consequences.
2. Preventing an Overgrowth of Biofilm
Our MetJ mechanism represses SAR11 biofilm production as bacteria population increases on the coral surface. This ensures that our biofilm will not suffocate the coral. For more information, visit our Project Description
3. Preventing the Spread of Our Engineered Bacteria
Our Biosafety construct drew inspiration from Johns Hopkins 2021 [6] — they utilized MATLab SimBiology Model to simulate their Biosafety construct — and found an antitoxin system that works for SAR11 bacteria. To prevent our engineered bacteria from forming biofilm in unwanted locations, we implemented a biosafety killswitch. This system relies on quorum sensing and operates by eliminating bacteria at low concentrations while allowing their survival at high concentrations. Under low bacteria concentrations, toxins are produced inside the bacteria; under high bacteria concentrations, toxin production is repressed. Simultaneously, small amounts of anti-toxins are generated to neutralize remaining toxins in high bacteria concentrations.
This strategy enables us to target and eliminate bacteria when they leave the coral, typically present in small numbers, while enabling coexistence with the coral when their populations are large. Importantly, toxins and antitoxins are exclusively produced within individual bacteria, ensuring no harm to the coral holobiont or other microorganisms. For additional information, please visit our Project Description
References
[1] Associate Professor Hsuan-Chen Wu. (n.d.). Dept. Of Biochemical Science & Technology, NTU. https://www.bst.ntu.edu.tw/ntubst2019EN/News_Content_n_41872_sms_46368_s_65964.html
[2] 實驗室生物安全教育訓練資訊. (n.d.). https://www.cdc.gov.tw/Category/MPage/5PdnFt4hFcFaw2eaJus9BQ
[3] Sen-Lin Tang. (n.d.). https://biodiv.tw/pi-Sen-Lin_Tang
[4] Prof David Bourne - Research Portfolio - James Cook University. (n.d.). jcu.me. https://research.jcu.edu.au/portfolio/david.bourne/
[5] Dr Lone Høj. (n.d.). AIMS. https://www.aims.gov.au/about/our-people/dr-lone-hoj
[6] Team:Hopkins/Model - 2021.igem.org. (n.d.). https://2021.igem.org/Team:Hopkins/Model