Team:Hong Kong-CUHK/Laboratory/safety

Safety Proposal

Recently, the outbreak of the new and fatal form of E.coli has awakened the public awareness on the safety of handling bacteria. This makes us design our safety strategy more seriously and thoroughly. When it comes to laboratory work,safety should always be put in the first priority. Otherwise, the laboratory practice could never be sustainable. Furthermore, we concern about not only the individual safety of the researchers, but also the public, the environmental safety, and even the safety issues for the future iGEM teams

A. Safety issues of our Project

Our project needs to express a light-driven ion pump, halorhodopsin, into E. coli. Thus, the bacteria can uptake chloride ions into the cells under light and the solution salinity varies. We demonstrate two advance usages of this protein. First halorhodopsin couples with P_gad, which is a chloride-sensitive promoter so that gene expression can be regulated by light. Second, we manufacture a pair of electrodes in order to generate electricity by combining sunlight, salinity difference together with the genetically engineered bacteria. Regarding to our project, we considered several safety issues that possibly be raised.

1. General biological safety

With regard to our researchers' safety, we have several precautions to ensure our team members can work in a safe condition. First and foremost, researchers have to attend laboratory safety workshops held by University Safety and Environment Office before they could join any laboratory work. The workshops cover mainly 4safety aspects, including general, chemical, radiation and biological safety.Also, the researchers have to wear gloves and laboratory coats throughout the laboratory work to avoid contacting any harmful, irritant, toxic or even carcinogenic reagents. Moreover, our project as well as the laboratory work is supervised by three professors together with three instructors who got Biological safety level Ⅱ certificate. Our researchers have also received biological safety level I certificate.Furthermore, our laboratory is a registered teaching laboratory with level Ⅱ safety which is suitable for bacterial and cell culture use.Inside the laboratory, students operate with autoclaved materials and follow cleaning procedures every time. We clean benches with 70% ethanol before and after we perform experiments. Last but not least, all the students maintain a good biological safety hood throughout the project in order to reduce any risk factor to the minimum.

2. Public and Environmental Safety

Firstly,we followed the regulation of the Hong Kong Government to purchased bacteria.Regarding to the bacteria we used, we chose 2 non-virulent strains of E.coli:DH5a for amplifying BioBrick and BL21 for expression of protein. These are to avoid putting public and environmental safety at risk. We follow safety rules so as to prevent harmful gene from leakage. DNA we use has also been sequenced and verified before use. And we disinfect all the cultures and wastes by autoclaving and bleaching before disposal to avoid leakage of any other potential risky substances e.g. genetically engineered bacteria. Finally, we promote synthetic biology and BioBrick to the public so more people can get familiar to this research area, and understand the importance of research safety. Through combining the above methods, therefore, we believe our project would neither cause any harm to the public nor the environment.

3. BioBricks

To answer the question "Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?" We believe our answer is No, the BioBrick parts we made this year are not going to raise any safety issues. The halorhodopsin is not toxic and exists commonly in halobacteria. It is also observed not to be infectious and pathogenic. So, the BioBricks we are going to make this year are not risky.

4. Antibiotics resistant plasmids

There is a concern that using antibiotic containing plasmid for transformation selection may possibly lead to the production of drug-resistance bacteria. The way of avoiding leakage of genetically engineered bacteria should also be considered.We make sure that every time we discard bacterial culture, we process them with30% bleach together with autoclaving procedures to reduce the probability of leakage to the minimum. Moreover, we transform bacteria with one single type of antibiotic every time, so as to ensure bacteria can only achieve resistance to one kind of antibiotics. Therefore, using other types of antibiotic can still kill the bacteria to stop the spreading.

5. Fabricating Eletrodes

Firstly,all our team members involved in handling of fine powder, such as graphite and sodium manganese oxide, are required to wear surgical or N95 mask in order to avoid the risk of irritation. Also, the team member responsible for the key steps in making the electrode has received formal training for working in the Nano Fabrication Laboratory and has a good understanding of the potential risks involved. Furthermore, we noticed that there is a small risk of cut involved in fabricating the electrodes into desired shape, where sharp tools like jigsaw may be used. In response to this, we decided at least one other person must be present for surveillance and reminding the working person. And a tidy working environment with good lighting and first-aid kit readily available will be provided for mechanical work.

6. Electric Circuit

There are common potential risks for short-circuiting, overheating and electric shock when it comes to using electric circuits. With regard to this, the circuit that we chose is equipped with modern safety mechanisms such as overload, overhearas well as short-circuit protection. To add on, the production of electric circuits involves the use of harmful chemicals, risk of burns in soldering, and cuts due to sharp points at the bottom of the circuit board. However, the teammembers responsible for designing and fabricating the electric circuits havereceived professional training in this aspect, and have prior experience in making electric circuit.

7. Risk Assessment and Managment

Besides individual precautions taken by our team in response to specific types of risks faced in different procedure, our team would also like to take a more systematic approach in minimizing the risk in our work. That is, risk management. We would like to bring forth the approach used by insurance andfinancial companies to laboratory safety as well. Classical theory of risk management involves aspects such as evaluation of risk, transfer of risk,dilution of risk, and reduction of risk.

• a. Evaluation of risk

As the name implies,before performing laboratory work, we think about the potential risks involved.This is done in a systematic fashion using a checklist. For example, our checklist asks what chemicals after involved in a protocol; is naked firerequired; is lifting heavy goods needed?. The hazard of a particular protocolis established. By making reference to the reports of past laboratory incidentskept by the University Safety & Environment Office, together with the formula Risk=Harzard x Probability, we derive how risky a particular procedurewould be. For our genetic engineering device, as suggested by the iGEM Headquarter, fault tree analysis is used to explore the consequences shouldmutations arise. The fault tree is constructed by branching into different possible means our bacteria could mutate, and after the first mutation,branching further to illustrate the effect of second and subsequent mutations.Fault tree allows easy estimation of risk using the above-mentioned formula.

• b. Transfer of risk

A well known principalin economics is “Principal of Comparative Advantage”. This theory applies well in risks too. Certain procedures may be done with less risk by a different party. For example, in fabricating our nano electrodes, it would be less risky if done by professionals specialized in material sciences compared to abiochemistry undergraduate. Hence we are considering outsourcing this part of research by purchasing the electrode or pay a service fee for the right professionals to make it on behalf of our team.

• c. Dilution of risk (also known as “Sharing of risk”)

It is obvious that lifting the very heavy water tank in our lab by two people would be less likelyto cause a sore back than just one. This demonstrates the “dilution of risk”.If the risk of a task can be reduced when more parties are involved in the task, the risk is said to be diluted. Another good example of “risk dilution”is always having more than one people to work in the lab, so that when emergency arises, there is redundancy to handle it. However, “dilution of risk”is actually trading between reducing overall risk, at the expense of puttingsomeone who is not at risk originally at a small risk. Therefore, careful planning is required before using this trick.

• d. Reduction of risk

Sometimes risks are avoidable, by making use of alternative procedures, reagents or equipment.Examples include:

- The risk of cuts can be reduced by using plastic instead of glass containers.

- The risk of cancer canbe cut by staining gels using GelRed instead of Ethdium Bromide.

- The risk of electric shock can be reduced by using a gel tank with cover at its top.

For “reduction of risk”,all one needs to do is simply be cautious. The attitude of team members is also important, as it would be a completely different story if these measures of risk management cannot be fully implemented.

In particular, two sets of risk assessment(General laboratory safety risk assessment and Biological laboratory safety risk assessment )  specific for our laboratory and project has been done by our instructors, so that we can have a more precise planning on choose of materials and reagents, division of labor and response to emergency etc.

The following is the hyperlinks to our assessment reports:

B. Local Biosafety Group at the Institution

The Chinese University of Hong Kong has a specific group, named University Safety& Environment Office. The office works for guidelines to all the facultiesfor safety issues, for example, laboratory safety, public safety, etc. Special guidelines which we strictly follow are given to us for handling microorganismsin the laboratory.

C. Suggestions to future iGEM teams

We suggest future iGEM teams not to manipulate any infectious or virulent bacteria in the project, so as to avoid any chance of causing harm because of executing the project idea. Also, we advice we should not use any harmful gene in the project. As chances of gene leakage are not possible to reduce to zero, usage of harmful genes should be avoided. We also have suggestions on making safer parts, devices and systems through biosafety engineering. For parts, we screen sequences with centralized database such as blast to detect if there is any virulent gene inside the part DNA. For devices and systems, we suggest theymust be regulated rigorously by promoter and terminator, so that the devices or systems can be switched off completely and conveniently to get an easy-managed controllable system. Below are some other suggestions to the future iGEM teams.

• Implement our “Risk management” approach
• Implement CUHK’s last year’s iGEM team work for labeling genetically-modified organisms to ensure traceability (on the origin and the information on the genetically-modified DNA).
• Use non-antibiotic selection to reduce the risk of super germ due to multiple antibotic resistance.
• Advocate for complete elimination of ethdium bromide, which was a commonly used mutagent for labeling DNA, by doing so, in newly established lab, there is no need to mark “EB area” etc.
• Advocate for chromosomal integration of engineered part of DNA to avoid horizontal gene transfer.
• Integrate a visible reporter (eg: GFP reporter) in the genetically engineered bacteria so contamination or spread of engineered bacteria in the environment can be identified quickly with a handheld UV lamp. GFP can be served as a common signal to indicate the presence of GM organisms.