Safety

Introduction

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The Nanyang Technological University (NTU) iGEM team views safety as one of the fundamental aspects towards experimental success. Our team is fully compliant with iGEM’s and NTU’s safety regulations – including chemical safety, biological safety, and data protection. Our approach towards safety follows two perspectives – individual and experimental safety.

For more details, please refer to our safety form instead.

Individual Safety

Indiviudal safety is further segragated into Individual Safety is further segregated into two categories – personal and laboratory safety.

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Personal Safety

Personal safety is of utmost importance as it directly protects the individual when he or she is conducting experiments. NTU Occupational Safety and Health (OHS) Management has mandated that all students involved in laboratory research are to complete various comprehensive risk assessment modules before embarking on their projects. This myriad option of modules available equips students with sufficient laboratory safety knowledge, and to conduct experiments in a safe manner. Each module has a 3-year coverage, and covers chemical safety, biological safety, and data protection:

OHS2BST01: Basic Safety Training

OHS2PCT01: SGSECURE Prepared Citizen Training

OHS2RMM01: Risk Management Core Module

OHS2CST01: Chemical Safety Training

OHS2DDR01: Donning a disposable respirator

OHS2HMR01: Fitting a half mask respirator

OHS2IR01: Working with Ionising Radiation

OHS3NIR01: Working with Non-ionising Radiation

OHS2SIG01: Understanding Signage from SS508

OHS2BBL01: Basic Biological Safety Training Course Module 1

OHS2BBP01: Basic Biological Safety Training Course Module 2

In addition, a risk assessment is required before embarking on a scientific research project. This allows students to identify any potential hazards, evaluate the risks involved, as well as develop additional mitigation steps to minimize the risk associated with any experimental procedures. When the Risk Prioritisation Number (RPN; given as Severity (S) x Likelihood (L)) exceeds a limit, additional risk control detailing the mitigation steps are required.

Table 1: Risk Assessment Table

No.	Work Activity	Hazard	Sub Hazard	Possible Accident/Ill Health & Person-at-Risk	Existing Risk Control	S	L	R	Additional Risk Control	S	L	R	Follow Up by & date
1	Use of chemicals	Chemical	Corrosive	Eye injuries and skin irritant	All personnel should wear proper PPE including lab coat, gloves and goggles. Safety eye wash and shower should be in working condition and easily accessed. Chemicals should be used in small quantities as practical and should be properly stored after use.	2	2	4	NA
			Sensitizer or irritant	Burns / scalds	All personnel should wear proper PPE including lab coat, gloves and goggles. Chemicals should be used in small quantities as practical and should be properly stored after use.	2	2	4	NA
			Poison	Chemical poisoning / absorption of chemicals or hazardous substances	All chemical containers should be clearly labelled with correct GHS labels. No food or drink is allowed in the lab.	2	2	4	NA
2	Use of machines	Electrical	Contact with electrical energy	Electrocution	Users must be careful when handling the equipment. Users should not touch the equipment directly while hands are wet.	5	1	5	NA
3	Working in coldroom	Physical	Extreme weather	Cold temperature environment	Users are required to attend a safety briefing for the coldroom before security access is granted. Users are told to dress properly such as wearing jackets, long pants etc before entering coldroom. Users are told not to work long hours in the coldroom and take frequent breaks every 30min. Users need to have a buddy system before working in the coldroom. Their buddies should check on them once every hour.	4	2	8	Users are required to have a buddy system before working in the coldroom. Their buddies should check on them once every hour.	4	2	8	Follow up by CCEB staff.

Laboratory Safety

The laboratory is also a certified Biosafety Level 2 (BSL-2) laboratory that is well-equipped with the appropriate safety features required for the project.

Biosafety Cabinets

Chemical Fumehood

Chemical Storage Cabinets

Eyewash Station and Shower

First Aid Kit

Experimental Safety

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All organisms used in our project are included in iGEM’s White List, ensuring they meet established biosafety standards. No additional check-in forms were required for non-White List organisms or parts, as none were utilized in our work.

Our experimental setup is also designed to minimize the risk of increasing antimicrobial resistance in our organism. The project focuses on exploring T7 RNA polymerase (T7RNAP), an enzyme derived from the T7 bacteriophage¹. Previous studies have characterized T7RNAP using an in vivo dual plasmid selection system, where chloramphenicol resistance serves as the readout^2,3. In these setups, better-performing T7RNAP variants conferred increased antibiotic resistance to bacteria, enabling them to thrive at elevated chloramphenicol concentrations. Using antibiotic resistance as a qualitative readout accelerates the identification of superior variants, as only bacteria harboring these variants survive under more stringent conditions. However, this approach raises concerns about unintended horizontal gene transfer, which could spread antibiotic resistance to other organisms. Such an event poses a potential biosafety risk, particularly if the organisms are not properly contained or disposed of.

Therefore, to avoid increasing the antibiotic resistance properties of our organism, we designed the experiment to quantify fluorescence intensity instead. Better-performing variants are expected to yield higher fluorescence readouts, enabling us to identify improved variants without relying on antibiotic resistance as a marker. This approach mitigates the risks associated with increased antimicrobial resistance while maintaining the efficiency of identifying superior variants.

References

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¹ Borkotoky, S., & Murali, A. (2018). The highly efficient T7 RNA polymerase: A wonder macromolecule in biological realm. International journal of biological macromolecules, 118(Pt A), 49–56. https://doi.org/10.1016/j.ijbiomac.2018.05.198

² Ikeda, R. A., Ligman, C. M., & Warshamana, S. (1992). T7 promoter contacts essential for promoter activity in vivo. Nucleic acids research, 20(10), 2517–2524. https://doi.org/10.1093/nar/20.10.2517

³ Boulain, J. C., Dassa, J., Mesta, L., Savatier, A., Costa, N., Muller, B. H., L'hostis, G., Stura, E. A., Troesch, A., & Ducancel, F. (2013). Mutants with higher stability and specific activity from a single thermosensitive variant of T7 RNA polymerase. Protein engineering, design & selection: PEDS, 26(11), 725–734. https://doi.org/10.1093/protein/gzt040