Team:KEYSTONE/Safety

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• Introduction
  • General Lab Safety
    • Behaviors
  • Prevention
  • First Aid Kit
  • Further Prevention
  • Experimental Safety
  • General Device Usage
  • Project Related Safety
  • Clarification
  • Project's Application Safety
  • Application
  • Potential Dangers
  • Potential Dangers’ Prevention
  • First-Aids

Introduction

As a synthetic biology team, we clearly recognize the significance of the safety of our project and prioritizes it before any other parts or results of our experiment. Our experiment utilizes the safest bacteria that could be used in biological labs of all: E. coli, while all our experiments were conducted under the reassurance that the environment, equipment, and operations are completely safe. Furthermore, we have ensured that our product would have no safety issues once entering the market. We organize our safety page into five sections: general safety, experimental safety, project application safety, and project application protections.

General Lab Safety

Behaviors

For this year’s project, our team follows the regulations strictly, which are classified as biosafety level 1 (BSL1). All the team members have received detailed lab trainings and are required to achieve a merit on the exam of lab safety before entering the lab. When our team is in the lab, participants’ behaviors are carefully regulated.

Fig. 1. The Lab Safety Precautions

The laboratory requirements include but are not limited to the rules listed below:

1. It is forbidden to turn the pipette tips, conical flasks, test tubes and other containers toward yourself and others’ faces to avoid liquid splashing

2. Before starting the experiments, check the experimental environment and reagents that need to be used in advanced. If it involves the use of odorous reagents (such as chloroform, isopropanol, etc.) you need to wear a mask in time, and ensure that the experimental space is ventilated and kept air flowing

3. Experimental equipment, pipettes, and various reagents on the contaminated area (operating table for protein electrophoresis and nucleic acid electrophoresis) should not be taken to other areas. Gloves that have been in contact with this area should be thrown away directly, and other experiments should not be directly touched area

4. Wear goggles and protective gloves before using the liquid nitrogen tank and place the operating procedures next to the liquid nitrogen tank.

5. The waste liquid (not exceeding 85% of the volume) and empty reagent bottles are labeled with orange waste labels and placed under the warning sign (big triangle skull logo)

6. The instructor of the biological project shall prepare a safety inspection log, and timely update and inspect the fire-extinguishing facilities of the laboratory, such as fire extinguishers.

Fig. 2. The lab safety exams we took

Prevension

First Aid Kit

The first aid kits are placed at the corners at our laboratory, with all the required materials, medicine, and equipment in them. The medicines within the first aid kits are replaced regularly by the supervisors in the lab to maintain their effectiveness. All the materials are reassured to be effective in a timely manner just in case of an urgent situation.

Further Prevention

There is also equipment for safety prevention placed around the lab, e.g., eye washer, fire extinguisher, blankets, lab coats, and goggles. All members in our team were educated and trained on the proper usage and circumstances. All prevention above can reassure that experimental accidents do not occur, and even if such accidents happen, every individual in the lab has the ability to handle the emergencies.

In order to prevent cross-contamination, our team made sure the experiments were done in aseptic technique when necessary. The laboratory equipment and material that has an aseptic requirement or environment are also guaranteed to be properly used.

- Large Centrifuge
The main task of this instrument in our experiments is to separate our whole bacterial solution into the supernatant and the sediment at a specific temperature (usually 4 degrees) at 12,000 mps. But to operate this apparatus safely, we need to pay attention to the following things: all tubes need to be leveled in pairs before entering this apparatus; all tubes with the same weight must be placed in the opposite holes of each other; the caps of the tubes need to be tightened; after leveling and placing, the inner caps need to be tightened and the outer caps need to be tightened.

- Ultra clean table
This apparatus is the place where we mainly do important experiments such as construction. The main purpose of this apparatus is to keep it in a sterilized state when not in use and to ensure a near-sterile environment. To operate this apparatus safely, we need to pay attention to the following matters. When you start to use this apparatus, make sure that all the UV lamps are off; make sure that all unneeded materials are placed to the side of the ultra-clean table (where they do not interfere with your operation); when you are finished using the apparatus, throw away all the non-reusable trash except for the reusable trash such as the spent gun head, which can be thrown in the trash can inside the ultra-clean table, and keep the ultra-clean table neat and clean.

- Shaker
This apparatus serves a function in our experiments to cultivate and promote the growth of bacteria at a fixed speed and temperature. In order to use this instrument safely, we need to pay attention to the following points: if you open the groove of the built-in fixed shaker, make sure that the groove is tightened and fastened when you finish using it; make sure that the conical flask is completely and safely locked when you place it in the mesh hole before you start the machine; make sure that the UV sterilization function is off when you place the conical flask in the shaker; be careful when you take out the locked conical flask. Do not break the glass bottle. If you break a glass vial, get help from the teacher in charge immediately, or wash your hands and arms where they are exposed or splashed with the solution. Afterwards, sweep out the glass scraps little by little and throw them into the glass scraps bin. Be careful of sharp objects during this time.

- Ultrasonic crusher
This instrument plays a role in our experiments to forcefully crush as well as separate the excess items of whole bacteria in the supernatant capacitance. When using this instrument safely, a few points need to be noted. When using the ultrasonic crusher, pay attention to the gun head when in the middle of the solution in the lower position to ensure the normal operation of the instrument; pay attention to the water level line in the 1L container where the ice is placed, preferably not more than 200ml; pay attention to the gun head not out of the centrifuge tube when in use.

- Electrophoresis apparatus
This instrument plays a role in our experiments to provide the detection of the quality of each sample. When using this instrument in a safe way, you need to pay attention to the following points. The gel inside the electrophoresis instrument needs to be placed in the recovery gel tank immediately after use, not to be touched directly by hand, you need to wear gloves; the gel should be recycled when using the whole film, not to be discarded at will; confirm the current voltages (usually around 100 volts) and the alignment of positive and negative poles when using the electrophoresis instrument; the length control of running gel, it is best to confirm again and again, usually in about an hour. Remember to wear protective equipment, such as gloves, protective clothing, etc., when operating and cleaning the instrument.

- Oven
This instrument plays the role of culturing bacterial solution in our experiment. When using this apparatus safely, you need to pay attention to the following points.When using the oven, make sure that the built-in space is completely empty and that there are no other centrifuge tubes placed inside for a long time; when using the oven, make sure that the temperature and the time of day are correct and that the tubes are not overheated or heated for a long time; when taking out the tubes, make sure that the instrument is completely cooled or mostly cooled before taking it out; when placing the tubes, make sure that all the lids are tightly closed; when operating the instrument, make sure that the protective equipment is worn. For example, gloves, lab coat, etc.

- Dissolved oxygen meter
This instrument serves as an instrument to test bacterial activity in our experiments over 10+ hours. Safe operation of this instrument requires a few points of attention. When operating the instrument, make sure that there are no errors in the connection between the probe and the instrument, avoid errors and broken wires, make sure that all probe wires are properly and tightly connected to the instrument; make sure that the centrifuge tube is in good condition when the probe is inserted, and that the bacterial solution is in good condition; after inserting the probe, make sure that the probe is tightly connected to the centrifuge tube and that more than one loop of sealing tape is used to After the probe is inserted, make sure the probe is tightly bonded to the centrifuge tube and more than one loop of sealing tape is used to completely wrap the gap between the tube and the probe; confirm the detection time, which is often not too long; after confirming the detection time, place the liquid in the probe upright on the centrifuge tube rack; after the detection is finished, make sure to wear protective equipment, such as gloves, protective clothing, etc. when handling the liquid.

- Enzymatic standard
This instrument plays the function of detecting the concentration of the constructed samples in our experiments. Safe operation of this instrument requires attention to the following points. When operating this instrument, all items, even the spot samples need to be held lightly to ensure that the lens is intact and will not break; the lens part of this instrument is relatively sharp, so be careful of sharp corners to cut the skin, so you need to wear good protective gear, such as gloves, protective clothing, etc.

Experimental Safety

General Device Usage

All of the devices that are common in a biological laboratory were taken good care of and used for suitable purposes. The pipettes, the 4 Celsius refrigerator, -20 Celsius refrigerator, vessels, centrifuges, the ice maker, etc., are all used with proper precautions, knowledge, and operation.

Project Related Safety

Clarification

1. There was completely no animal usage at all throughout our project

2. No special bacteria with potential dangers were used

3. There is an extremely low potential risk of creating super bacteria. All of the antibiotics were applied carefully, and we selected the antibiotics cautiously, making sure that even if experimental accidents occur, the risk of creating super bacteria is low.

Project's Application Safety

Application

If our product enters the market, then it would be used within factories to perform the role of the biodegradation of rubber. Our team has two aims: to both publicize this technology, and to further enlarge its effectiveness through mass. In order to achieve the two goals, we have designed two fermentation methods for Lcp. We focus on the continuous fermentation of Lcp on a smaller scale with more convenience to enlarge the range of suitable audiences. On the other hand, we would adapt batch fermentation of Lcp on a larger scope, to make sure this technology could be used within factories for mass biodegradation.

Potential Dangers

There are no major potential dangers led by our product. We have used the safest proteins and bacteria throughout the entire project. We used E. coli as our bacteria, and inserted Lcp protein, NusA protein, and HlyA signal peptide into our plasmid. Based on research, all of the materials used do not produce toxicity toward human body.

The biggest risk that could happen for the customers is when they accidentally ingest E. Coli, which might lead to diarrhea, stomach cramps, and fever for sensitive bodies. However, this situation is not likely to happen since such devices would be reserved within environments where the customers have sufficient knowledge to operate the devices.

Potential Dangers’ Preventions

In order to prevent the potential risk, we have adopted some prevention methods. Instructions about the danger of accidental ingestion of E. coli would be assured to be provided to the customers before they utilize the product. Such reminder and precautions could ensure that the customers would not encounter such potential risks.

Furthermore, public education on the proper usage of the devices could also assist the process of prevention. By warning the public on such dangers when using the product, the targeted audience (medical workers) would be cautious of the forbidden operations when having contacts with the device.

Hence, such potential risk could be prevented.

First Aids

Even if the customers accidentally ingested E. coli from our product, first aids could be provided to ensure their safety. Since our targeted audience are medical workers, the first aid preparation becomes relatively easy. Our team have to make sure that if this product is applicated in reality, the customers have the basic first-aid kits around them and have the circumstances and ability to provide medical support for such emergencies.

By doing so, we would have a double-layered protection for the customers, guaranteeing that even if potential accidents occur, it could be solved.