Team:Technion Israel/Practices/safety
Safety
Safety is a top concern in the lab and in all food, health, and cosmetics products. Throughout our project, we always kept safety in mind, especially since the product we are developing may influence the microbiome of the scalp for the user. Here are some of the ways we kept safety on our minds:
Lab Safety
Our team works in a laboratory classified as having Biosafety level 1, which is the lowest, applying to work with microorganisms that pose minimal potential threat to humans. The risks we encounter are common in every molecular biology lab, and can be easily minimized through compliance with a strict code of conduct, largely similar to World Health Organization (WHO) Laboratory biosafety manual 1. Here we will review some of the main precautions taken.
Biological waste treatment is of great importance due to possible risk of GMO release to the environment, and release of bacteria containing self-replicating plasmids with antibiotic resistance. We took the following precautions in order to avoid the spread of those genes: all disposable equipment in possible contact with laboratory microorganisms (tips, petri dishes, gloves, etc.) were collected in biohazard bags and autoclaved before disposal, liquid media was sterilized with 96% ethanol overnight before their containers were washed. We always wear gloves and lab coats while handling such materials and open doors while wearing gloves by grabbing the handles only in areas marked specifically for touching with gloves.
Personal Safety
Each Technion student has to receive safety training before starting a project involving wet lab work. The aim of the training is to learn about safety measures and proper use of equipment in the lab. In addition to this, all iGEM team members participated in a nano-course - two weeks of laboratory training in genetic engineering and cloning techniques, with emphasis on safety and proper laboratory techniques, given by experienced personnel. The key to safe and efficient lab work lies in safety-conscious and responsible implementation of guidelines.
Work with bacteria sometimes requires minimizing the distance between the person handling the bacteria and the bacteria itself (making starters, plating cells, inoculating bacteria, etc.). To avoid the risk of aerosols, as well as cross-contamination, we worked near fire when handling bacteria, using disposable loops and plate spreaders.
Many molecular biology techniques involve chemicals which pose a danger when inhaled or when coming in contact with the skin, requiring additional safety measures. For example, Ethidium Bromide is a known carcinogen, used for DNA staining in agarose gel. Therefore gel preparation was done in designated marked areas, addition of EtBr was done in a hood, and special emphasis was placed on wearing gloves, lab coats and covering the face when cutting bands from gel using UV.
Genetically modified microorganisms
All bacterial strains used during our project were risk group 1 organisms:
- Bacillus subtilis PY79
- Escherichia coli
- DH5α
- BL21
- TOP10
- MG1655 Wild-type
- MG1655 Δpgi ΔUdhA
Parts
Our main part is the 3α-HSD enzyme from rat liver origin (AKR1C9), which also exists in the human body as four different isoforms (AKR1C1-4). In the prostate, 3α-HSD converts the potent androgen 5α-DHT into a less active form - the reaction in the heart of our project. In order to minimize the risk of hormonal imbalance, this reaction will take place topically, on the human scalp and will be catalyzed by an isoform specific as possible 2.
The second half of our project focuses on NADPH overproduction. NADPH is an essential cofactor for our desired reaction. It is an abundant reducing agent, which isn't considered dangerous even in highly purified solutions, according to the Sigma-Aldrich MSDS.
Product safety
Our final product will include two separate containers, one containing B. subtilis secreting our enzyme, and the other containing the E. coli overproducing NADPH. A contraption consisting of a syringe and a comb (see the design section of the wiki for more details) will be used to apply the B. Subtilis bacteria onto the scalp. The other compartment, containing the E. coli strain, will be equipped with a filter which will keep the bacteria and large molecules in the solution inside the container, while allowing the NADPH to pass through onto the scalp. Direct contact between consumers and genetically modified bacteria raises several safety challenges which must be addressed.
Consumer exposure
Naturally found on our skin and scalp, our organism of choice is a wild type B. subtilis. The strain is commercially used as a skincare product, a food ingredient for human consumption, in animal feed, in fertilizer, and in an antibiotic substitute 3. Its widespread use hints at its low-risk usability in commercial products. Even though both B. subtilis and E. coli used in our product are poor colonizers and lack the ability of producing a significant amount of toxins to harm humans, we chose to keep the E. coli trapped in a membrane inside a special syringe, so only B. subtilis will come into contact with the user’s skin 4,5.
E. coli K-12 strains are considered enfeebled organisms after being maintained in a laboratory environment for more than 70 years. They have also a defective cell wall, so they lack the ability to efficiently recognize and adhere to the mucosal surfaces of the intestinal tract, contrary to other strains found as part of the natural microflora. Moreover, organisms grown under laboratory conditions in general are not particularly competitive in comparison to microorganisms isolated from the environmental niche of the organism 4.
As in any other skincare product, in our product, in some cases, an allergic reaction may occur. Therefore, our user manual will include warnings and will refer to possible incidents requiring the user to consult a doctor. The consumers will also be able to sterilize the containers and comb by themselves, using 70-85% alcohol supplied as part of the final product kit.
Genetically modified organism release to environment
Another advantage of working with B. subtilis is that genetic modifications are made in the genome itself, so the chances of gene transfer decrease due to the absence of independent replicating plasmid. Theoretically, our engineered B. subtilis is able to exchange genes through recombination with other closely related strains, though under most conditions the organism is not biologically active, rather exists in the spore form 5, thus limiting the spread of the antibiotic resistance genes.
In addition to this, survival of E. coli outside its natural habitat (mammalian bowels) is very limited and a spore form doesn't exist 4.
In both cases, for personal use the amount of cells that risks being released to the environment is not even close to the amounts released as result of industrial fermentation processes used for the production of a variety of proteins and enzymes, and considered of low risk level according to EPA (U.S. Environmental Protection Agency) 4,5.
Regulation
To make the final product even safer, we will use a kill switch designed for each organism to minimize the risk of release to the environment. For B. subtilis we plan to use a part developed by METU_TURKEY 2013 (BBa_K1197007). The kill switch turns on in absence of IPTG, an inducer of the B. subtilis promoter used in our project, so as the IPTG supplement is fully consumed, the bacteria dies.
In E. coli we would add the kill switch designed by the Technion-HS 2015 team, tying the bacteria to a specific medium containing AHL. The kill switch was submitted by the team: BBa_K1767003. The illustrated circuit can be seen in Figure 1.
1. Laboratory biosafety manual, 3rd edition, World Health Organization, Geneva 2004. 2. Penning TM, Jin Y, Heredia VV, Lewis M, Structure–function relationships in 3α-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms, Journal of Steroid Biochemistry & Molecular Biology 85 (2003) 247–255. 3. Gonzalez D.J, Haste N.M, Hollands A, Fleming T.C, Hamby M, Pogliano K, Nizet V, Dorrestein P.C (2011), Microbial competition between Bacillus subtilis and Staphylococcus aureus monitored by imaging mass spectrometry, Microbiology 157(Pt 9): 2485–2492. 4. Escherichia coli K-12 Derivatives Final Risk Assessment, U.S. Environmental Protection Agency 1997. 5. Bacillus subtilis Final Risk Assessment, U.S. Environmental Protection Agency 1997.