Team:ZJUT China B/Safety
Safety
As an ancient saying goes, "Prudence can catch cicadas for thousands of years, and caution drives a ship for ten thousand years. " The safety of the project is not only related to the success of our experiment, but also affects the environment and even our life safety. Therefore, safety is the top priority in our project.
General lab safety
It's really an honor that our iGEM team has an exclusive laboratory. Dr. Zhang Yinjun is the person in charge of our laboratory, who is responsible for the safety and security of our biology labs. In addition, Institutional biosafety officers will also supervise us, giving us safety training courses in the laboratory. They designated a special equipment-managing manager to inspect special laboratory equipment and gave special personnel certain certificates. The employed personnel must receive special training, and only after passing the examination, they can get their certificates. It is very clear that no work is allowed without certificates. Our Laboratories also need to establish special equipment operation files to ensure the safety of our experiments. All these experts are experienced in working with organisms or parts that might be risky. And they are also familiar with the experimental procedures and practices we will do.
Safety education
Before entering the labs, under the organization of our instructors, we learned an online course called "laboratory access" and an offline course called “the Detailed Rules of Laboratory Safety and Environmental Protection Supervision, Assessment, Rewards and Penalties” formulated by the school.
We have also developed our own laboratory guidelines and strict requirements for our team members:
1.You must wear lab uniform before entering the lab, which means you can't do experiments without them.
2.Do not wear barefoot shoes and better wear long trousers. Pay attention to personal protection.
3.Girls should tie up their hair;
4.Wear gloves during the experiment;
5.No eating in the laboratory;
6.Use toxic reagents carefully without touching the skin, especially wounds;
7.Take things out of the oven in time;
8.Pay attention to safety during the experiment. If the glassware is broken accidentally,
you must be careful with the disposal of fragments in case of cutting your fingers;
9.The pipette gun should be adjusted back to the maximum range after using；
10.Everyone should mark his/her own things (name + number + date + name) and be responsible for his/her own things;
11.After the experiment, the glassware should be cleaned and placed neatly, and the used stuff should be returned to
their original places;
12.Clean the used media in time and after sterilization too;
13.All garbage shall be treated uniformly and not be discarded at will;
14.Pay attention to laboratory hygiene;
15.The last person to leave the laboratory should remember to check the equipment, hygiene, lights and doors.
And shut down the ultra clean table;
Instrument safety
Our laboratory has equipment which meets the primary and secondary biosafety standards. And the instruments and equipment used in this project include Centrifuges, Electrophoresis System, Spectrophotometer, PCR Amplifier, ELIASA, Chromatographic columns, Shaker, Vertical Clean Benches, Electric Sterilizer, AKTA Explorer, Constant temperature incubator, Freeze Dryer. All instruments and equipment are checked regularly, maintained in time and cleaned before and after the experiment to ensure the accuracy and reliability of the experimental results. Wet lab members who participates in the experiment will receive appropriate training before using the instruments and they will also learn the correct operation of the instruments according to the instructions. Any experiment will be carried out with at least one person present. For the instruments commonly used in the laboratory, the team members sorted out and listed the notes, some of which are shown as following.
Vertical Clean Benches:
- Don't do experiments when the ultraviolet lamp is on.
- Keep the ignition source away from the ethanol to prevent an explosion while using the alcohol lamp and alcohol.
- Don't push the windshield up and down so hard in case of hurting someone.
Shaker:
- Check whether the base is firm to prevent the lask from toppling, which may cause rust, leakage, and fire.
- Set the temperature of the shaker so that it’s not too high to cause fire or too low to affect the propagation of bacteria.
- If there is any abnormal condition (burning smell, etc. ), the power plug should be removed immediately to turn off the instrument, or the power supply should be disconnected by the circuit breaker.
Electric Sterilizer:
- Check whether the base of the shaker is firm, and prevent the shaker from tipping over during shaking, which may cause rust, leakage, and fire.
- Don’t touch the upper cover of the sterilizer to prevent skin burn when using.
- When using the electric sterilizer, please be sure to wait until the pressure drops to 0 before opening the exhaust valve, so as not to cause the explosion.
- During sterilization, pay attention to the temperature, pressure and time.
- After sterilization, be careful of the overflow of the steam when opening the sterilization pot to avoid being scalded by steam.
Freeze Dryer:
- Remember to precool for 30 minutes or more before using it.
- Samples prepared by freeze dryer should enlarge surface as much as possible, and should not contain acid, alkali and volatile organic solvents.
Operational safety
1. When using the ultra clean bench, turn off the ultraviolet lamp, turn on the floodlight and ventilator, and do not perform experimental operation when the ultraviolet lamp is turned on;
2. Before and after the experiment, disinfect and wipe the working area with 70% ethanol or 10% fresh bleach, especially the area that may encounter relevant reagent or bacterial solution;
3. When using an alcohol lamp, do not ignite another alcohol lamp by using this alcohol lamp;When adding alcohol, be careful not to be less than 1/3 of the volume of the alcohol lamp, not more than 2/3 of the volume of the alcohol lamp;When the alcohol lamp is lit, people should beside it and turn off the alcohol lamp if they leave;
4. Do not put unnecessary items on the ultra-clean bench, turn off the floodlight and ventilator after use, and turn on the ultraviolet lamp again for sterilization;
5. All operations requiring sterility should be carried out beside the ultra-clean bench and alcohol lamp. Solutions or articles containing live bacteria should be sterilized and disinfected before unified treatment.
6. When using pipette to draw liquid, pay attention to replace the nozzle to avoid cross contamination of liquid;
7. Before and after coating or inoculation, the coating stick or inoculation ring should be burned on the alcohol lamp for sterilization;
8. Specimens shall be taken with forceps during microscope observation. Disposable specimens shall be cleaned and sterilized after use before unified treatment;
9. Wear gloves when using the dye EB during agarose gel electrophoresis and do not contact directly. The electrophoresis tank should be covered to prevent evaporation of liquid and reduce the possibility of electric shock.
10. Wear gloves to get things heated in microwave oven to avoid scald;
11. When using the centrifuge, pay attention to balance the centrifuge tube by weighing the balance, cover the centrifuge tuber, and do not open the hatch until the centrifuge stops completely;
12. Before using the autoclave sterilization pot, check whether the amount of distilled water in the pot is sufficient and check whether each valve and switch are normal;After sterilization is over, allow it to cool naturally before opening the exhaust valve for exhaust.
Project safety
All we have to do is testing virus. It sounds dangerous. Therefore, when we designed the project, we fully considered the issue of security. We carefully filled in the Safety form and the check-in form, and carried out experiments in strict accordance with the official requirements of iGEM. The whole project is completely free from any danger.
Virus mimics
In the absence of access to the virus and the fact that our laboratory does not allow experiments with live pathogens, we came up with a method to simulate the pathogen -- to intercept a small segment of characteristic sequence (about 80bp) of the virus to construct a plasmid and transfer the plasmid to E. coli BL21 and transcribe it into RNA, so as to obtain viral RNA for experiments. The virus sequence is not pathogenic and is safe and harmless.
Non-pathogenic strains
For the safety of the experiment, non-pathogenic strains were selected, including E. coli BL21, E. coli DH5alpha and E. coli Rosetta 2(DE3)pLysS. These chassis microorganisms are commonly used in the laboratory and cleaned after high temperature sterilization without any features that may endanger human and environmental safety. So, from the point of view of the strains we use, our project is safe.
Harmless plasmids
The plasmids we use in our project: pET-28a, pC013, pC0061, p2CT, etc. , are almost harmless to humans. Therefore, plasmids do not cause safety problems.
Device safety
Our team (ZJUT_China_B) has designed a multi-virus detection system based on Cas13 system. This is a user-friendly and affordable virus nucleic acid detector that can be used for home detection, medical diagnosis and other purposes. As a medical device, safety and effectiveness play a vital role in health care, because defective or unsafe medical devices may cause serious consequences. Our equipment does not contain bioengineered bacteria, only the lyophilization and buffer of related enzymes, so it has good biological safety.
Fluorescence detector
Our device is divided into two parts: fluorescence detector and reaction chip. The fluorescence detector is all electronic components, mainly including single chip microcomputer, light source, filter and optical sensor.
Reaction chip
Our reaction chip is designed for one-time use. It contains lyophilization and buffers of many related enzymes, and they are all sealed. The patient injects the sample into the chip and a series of reactions occur. Finally after the result is obtained in the detector, the chip will be treated as medical waste. During our verification process, no real virus samples will be placed in it. We will only place a small virus-related nucleic acid fragment to verify the feasibility of the device.
Safety in use
In order to prevent users from getting hurt and not using the product, we have the following plans:
1.In the outer package and instructions of the product, the "safe operation guide" is marked in the form of text and pictures respectively, and the method of correctly using "viralertor" is introduced.
2.Our products are very small, we need to indicate on the outer package that attention should be paid to parts falling off and avoid the danger of being eaten by children, and should indicate that they should be used under the guidance of adults.
3.Each of our products is equipped with disposable gloves and disposable swabs to make it safer for users to operate.
4.Due to the needs of product design, the biochemical reagents in our products are strictly sealed, and users do not need to worry about leakage under normal circumstances. If users are worried about leakage, we suggest heating at 60 degrees Celsius for 30 minutes or more for disinfection.
Research safety
When carrying out our human practices work and thinking about the implementation of our project, we also thought a lot about safety with regards to the moral duty we felt for our audience in engagement activities, users of our viralibrary database, and the environment and garbage collectors in the waste disposal related to our project. Please see our >> Ethics page << of our Human Practices pages for more information on this.
1. Wear a mask all the way on the bus or subway.
2. Wear a mask throughout the ride in a ride-hailing car or taxi. If the driver does not wear a mask, remind the driver of wearing a mask or change to another ride-hailing car.
3. Wear a mask and helmet throughout the cycling.
4. Obey the traffic rules and be cautious while driving, cycling or walking, .
5. Cover your coughs and sneezes with a tissue or sleeve.
6. Reduce touch on public stuff in public places.
7. Avoid touching your face with your hands when you are out.
8. In crowded places, a safe distance of more than one meter should be kept between people.
9. Try not to eat outside. Avoid direct contact with cutlery when dining with others.
10. Strictly obey the campus epidemic prevention rules and the daily self-temperature measurement.
11. After arriving home from outside, please wash your hands strictly according to the standard.
12. If you have been to high-risk areas or contact with people from these areas, please report in time and pay attention to your own safety and take protective measures.
References
[1] Max J. Kellner, Jeremy G. Koob, Jonathan S. Gootenberg, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nature Protocols, 2019, 14(10):2986-3012.
[2] Jonathan S. Gootenberg, Omar O. Abudayyeh, Max J. Kellner, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. 2018, 360(6387):439-444.
[3] Omar O. Abudayyeh, Jonathan S. Gootenberg, Silvana Konermann, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. 2016, 353(6299):aaf5573.
[4] Kyung-Min Kwon, Sung Gyun Kang, Tatyana G. Sokolova, et al. Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR. Enzyme and Microbial Technology, 2016, 86:117-126.
[5] Hui eYuan, Li ePeng, Zhong eHan, et al. Recombinant expression library of Pyrococcus furiosus constructed by high-throughput cloning: a useful tool for functional and structural genomics. Frontiers in Microbiology, 2015, 6
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