Team:Hong Kong-CUHK/Laboratory/safety

Safety Proposal

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

 

A. Safety issues of our Project

Ourproject needs to express a light-driven ion pump, halorhodopsin, into E. coli. Thus, the bacteria can uptakechloride ions into the cells under light and the solution salinity varies. Wedemonstrate two advance usages of this protein. First halorhodopsin coupleswith P_gad, which is a chloride-sensitive promoter so that geneexpression can be regulated by light. Second, we manufacture a pair ofelectrodes in order to generate electricity by combining sunlight, salinitydifference together with the genetically engineered bacteria. Regarding to ourproject, we considered several safety issues that possibly be raised.

 

1. General biological safety

Withregard to our researchers' safety, we have several precautions to ensure ourteam members can work in a safe condition. First and foremost, researchers haveto attend laboratory safety workshops held by University Safety and EnvironmentOffice 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 thelaboratory work to avoid contacting any harmful, irritant, toxic or evencarcinogenic reagents. Moreover, our project as well as the laboratory work issupervised by three professors together with three instructors who gotBiological safety level Ⅱ certificate. Ourresearchers 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 followcleaning procedures every time. We clean benches with 70% ethanol before andafter we perform experiments. Last but not least, all the students maintain agood biological safety hood throughout the project in order to reduce any riskfactor 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 toavoid putting public and environmental safety at risk. We follow safety rulesso as to prevent harmful gene from leakage. DNA we use has also been sequencedand verified before use. And we disinfect all the cultures and wastes byautoclaving and bleaching before disposal to avoid leakage of any otherpotential risky substances e.g. genetically engineered bacteria. Finally, wepromote synthetic biology and BioBrick to the public so more people can getfamiliar to this research area, and understand the importance of researchsafety. Through combining the above methods, therefore, we believe our projectwould neither cause any harm to the public nor the environment.

 

3. BioBricks

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

 

4. Antibiotics resistant plasmids

There is aconcern that using antibiotic containing plasmid for transformation selectionmay possibly lead to the production of drug-resistance bacteria. The way ofavoiding 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 ofleakage to the minimum. Moreover, we transform bacteria with one single type ofantibiotic every time, so as to ensure bacteria can only achieve resistance toone kind of antibiotics. Therefore, using other types of antibiotic can stillkill the bacteria to stop the spreading.

 

5. Fabricating Eletrodes

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

 

6. Electric Circuit

There arecommon potential risks for short-circuiting, overheating and electric shockwhen it comes to using electric circuits. With regard to this, the circuit thatwe chose is equipped with modern safety mechanisms such as overload, overhearas well as short-circuit protection. To add on, the production of electriccircuits involves the use of harmful chemicals, risk of burns in soldering, andcuts 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 inmaking electric circuit.

 

7. Risk Assessment and Managment

Besidesindividual precautions taken by our team in response to specific types of risksfaced in different procedure, our team would also like to take a moresystematic approach in minimizing the risk in our work. That is, riskmanagement. We would like to bring forth the approach used by insurance andfinancial companies to laboratory safety as well. Classical theory of riskmanagement 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, ourchecklist 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 theformula Risk=Harzard x Probability, we derive how risky a particular procedurewould be. For our genetic engineering device, as suggested by the iGEMHeadquarter, fault tree analysis is used to explore the consequences shouldmutations arise. The fault tree is constructed by branching into differentpossible 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 wellin risks too. Certain procedures may be done with less risk by a differentparty. For example, in fabricating our nano electrodes, it would be less riskyif done by professionals specialized in material sciences compared to abiochemistry undergraduate. Hence we are considering outsourcing this part ofresearch by purchasing the electrode or pay a service fee for the rightprofessionals to make it on behalf of our team.

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

It is obvious thatlifting 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 thetask, 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 whenemergency 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, carefulplanning is required before using this trick.

• d. Reduction of risk

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

- The risk of cuts canbe 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 electricshock 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 alsoimportant, as it would be a completely different story if these measures ofrisk management cannot be fully implemented.

 

In particular, two sets of risk assessment(General laboratorysafety risk assessment and Biological laboratory safety risk assessment )  specific for our laboratory and project hasbeen done by our instructors, so that we can have a more precise planning onchoose of materials and reagents, division of labor and response to emergencyetc.

 

The following is the hyperlinks to our assessment reports:

 

 

 

B. Local Biosafety Group at the Institution

TheChinese 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. Specialguidelines which we strictly follow are given to us for handling microorganismsin the laboratory.

 

C. Suggestions to future iGEM teams

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