Safety
Introduction
This year, all experiments and researches conducted by Tongji-China 2023 were in accordance with the safety policies of iGEM competition and lab safety rules of our nation and school. In the early stage of our project, we estimated possible safety risks in all aspects of our project and lab work, and then formulated guide lines and strategies for the safety problems we might come across. At the same time, there is a complete report and management system regarding emergencies, which can further guarantee the safety of lab personnel.
今年，Tongji-China队伍的所有实验工作都遵照了iGEM比赛的安全指导以及国家、学校的实验安全要求。在项目初期我们从各个方面评估了本项目实验部分可能存在的安全风险，并且针对可能出现的实验安全问题制定了相应的方案。同时，我们实验室还具有完善的紧急事件上报处理体系，为实验室人员安全提供了进一步保障。
Project design
Chassis of choice
When deciding on the chassis, we did plenty of research about which organisms can or probably can assemble the pilin from Geobactor metallireducans into conductive pilus(in other words, organisms that have Type Ⅳ pilin). Commercial E.coli strains Top10 and DH5α have been proven by other iGEM teams to be able to produce conductive pilus from Geobacter sulfurreducens, which also belongs to Type Ⅳ pilin. However, endogenous Type Ⅰ pilin in Top10 and DH5α must be repressed and a large cluster of Type Ⅳ pilin related genes must be introduced. Another E.coli strain, enterohemorrhagic escherichia coli(EHEC), has a full set of Type Ⅳ pilin system genes but it is highly pathogenic and can cause serious diseases. Considering the policy of iGEM and our own safety, we did not choose this strain although its T4P system suits our project well.
Vibrio natriegens is an emerging chassis in the field of genetic engineering and synthetic biology. V.natriegens is the fastest-growing bacterium reported so far and can produce a large mass of proteins in a short period of time. Also, after plenty of research, we found that V.natriegens has Type Ⅳ pilin system. Even if it is a new chassis organism, a lot of Genetic manipulation tools for V. natriegens have been proposed. So finally, we chose Vibrio natriegens as our chassis organism.
| Comparison | ||
|---|---|---|
| Strain | Pros | Cons |
| Top10, DH5α | Allows high-efficiency protein expression of any gene that is under the control of a T7 promoter and has a ribosome binding site; easy access and mature management methods. | Has no T4P and cannot assemble the Pilins properly. (A key gene in their Type Ⅰ pilin system needs to be knocked out and T4P genes need to be introduced.) |
| EHEC | Has T4aP and has been proven to be able to assemble the Pilins using relevant gene cluster. | Pathogenic. Cannot be handled under the biosafety level of our lab. Complicated operation procedure. |
| Vibrio natriegens | Has T4aP and can assemble the pilins into functional pilus; Rapid proliferation, able to double with a generation time of about 15 min; able to produce greater amounts of biomass and generate large amounts of proteins in a shorter time. | For Vibrio natriegens ATCC14048, chemical transformation and electroporation efficiency is lower than those of commercial E.coli strains. |
Parts of choice
Our project is based on the conductive pilus of Geobacter metallireducens and a supporting CRISPRi system. For the system to perform, we created or chose several parts. Before starting our lab work, we carefully checked the biosafety of the parts we had chosen. Recombined sfGFP was a merge protein of N terminus of m.PilA(PilA of Geobacter metallireducens) and sfGFP. sfGFP is a widely used report gene and is generally considered safe. PilA is the monomer that assembles into conductive pilus in Geobacter metallireducens. Type Ⅳ pilus is widespread in bacteria possibly because of their extreme functional versatility. Unlike many pathogenic bacteria with toxic or adhesive Type Ⅳ pilus, the conductive pilus of Geobacter metallireducens function in communication with other cells and Fe(Ⅲ) reduction which have not been reported harmful yet.
SpdCas9 is a catalytically dead mutant of the Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system. When sgRNA guides spdCas9 to the target sequence near a promoter, this complex can block the binding of critical transcription factors to the cis-elements, leading to repression of a specific gene. Streptococcus pyogenes is a human-specific pathogen that can cause a wide range of diseases. However, spdCas9 is a widely used safe genetic engineering tool and also a registered part in iGEM registry. The sgRNA we designed is all targeted at the promoter of PilA of V.natriegens and will not interfere with human genes.
Safe lab work
Laboratory Safety Regulations
1. Personal Protective Equipment (PPE):
- Always wear appropriate PPE, including lab coats, gloves, and closed-toe shoes when working in the laboratory.
- Ensure PPE is in good condition and properly fitted.
2. Chemical Handling:
- Be familiar with the Material Safety Data Sheets (MSDS) of chemicals used in experiments.
- Handle chemicals in a fume hood when necessary to prevent inhalation exposure.
- Label and properly store all chemicals.
3. Equipment Safety:
- Use laboratory equipment only if trained and authorized .
- Inspect equipment for defects before use and report any issues immediately.
4. Emergency Procedures:
- Know the location of safety showers, eyewash stations, fire extinguishers, first-aid kits, and emergency exits.
- In case of accidents or spills, follow the laboratory's emergency protocols.
Bacteria Handling Safety Guidelines
1. Containment:
- Work with genetically engineered organisms in designated containment areas.
2. Sterilization
- Sterilize equipment and materials before and after use.
- Periodically clean and sterilize the working area thoroughly.
- Containers contaminated by bacteria are sterilized by dry heat or 75% ethanol.
3. Disposal:
- Wastes contaminated by bacteria are harmlessized before diposal.
- Contaminated wastes are diposed and handled seperately from other lab wastes.