As with everything in life, there are always risks and dangers attached with doing lab work. Thus, we have done our best to minimize the safety risks by adjusting the environment, using non-threatening Biosafety Level 1 bacteria and strictly following the lab safety rules.

General Lab Safety

Our lab in the METU Biological Sciences Department was a BL-1 (Biosafety level 1) facility. BL-1 is the lowest safety level, indicating that it didn’t include any pathogens that could be harmful to us. Despite this low risk, we kept our guard up by always complying with lab safety rules, wearing appropriate clothing and by working in the hood at all times.

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Before we stepped into the lab, we were trained on biosafety rules and how to behave in the best manner to keep ourselves and other people safe and secure in the lab. We were also put through an examination that tested our knowledge on biosafety rules at the end of those lessons. After passing these exams, our lab experience has started. Our PI, additionally, prepared a presentation in which she highlighted all the important points and considerations the day we had our first lab experience along with making sure that we were always using personal protective equipment.

In the lab environment, we worked in a class II biosafety cabinet to avoid the contamination for both sides and acted extremely cautious in case of any hazards. We worked close to fire and tried to avoid contaminations.

The bacteria we have worked on, E.coli DH5⍺ and ethanologenic strain KO11, were respectively provided by New England Biolabs (NEB) and American Type Culture Collection (ATCC).

We, during our experiments, followed all the protocols supplied step by step, taking all the precautions and never conducted any experiments without the guidance and presence of our principal investigator and secondary instructor.

Safe Wetlab

After all the biosafety training we received about the lab environment, PPE and biosafety rules; we also planned all the experiments and lab days in advance to always be ahead of the curve, to make sure nothing would go wrong. Most of the methods we used were iGEM protocols, however, when necessary, we also used protocols that were more compatible with our bacteria. None of these protocols/methods included any chemicals that could be harmful to us/living tissues when inhaled or contacted.

Moreover, the disposal of the Petri dishes and liquid/solid wastes was checked constantly and done properly; with firstly being autoclaved and then placed in the biological waste bin.

Before and after each experiment, the working place and equipments such as pipettes, racks etc. were cleaned with alcohol and sterilized.

Safe Project

This low-risk level was consistent for all of our experiments as we used the Biosafety Level 1 E. coli ethanologenic strain KO11 and DH5⍺ (Ingram et al., 1998; U.S. Public Health Service Guidelines, 2009).

Our choice of parts were also non-pathogenic and harmless. The gene GSH is an antioxidant found in living tissues of humans and is an essential element in detoxification. (Pizzorno, 2014). Moreover, it is found in surprisingly high levels; the same concentration as glucose, potassium, and cholesterol (Pizzorno, 2014). Similarly, the FucO gene plays a role in detoxification by acting upon furfural and 5-HMF, turning them into non-toxic chemicals (Wang et al., 2011). In brief, we only used the non-pathogenic and harmless genes having the role on detoxification of toxic and threatening substances, posing no threat to human health.

In order to analyze our PCR products, we added loading dye with glycerol to our samples and ran them on the gel. However, in this protocol, we also had to use Ethidium Bromide. Ethidium Bromide is an intercalating tag, a nucleic acid stainer, but it must additionally be handled carefully and decontaminated prior to disposal. Its biggest drawback is that it is a potent mutagen. As we were aware of this information during our experiments, we made sure to wear a lab coat, mask, gloves which we discarded after working with EtBr (Thermo Fisher Scientific, n.d.).

Environmental Impact:

During these stages of our project, our bacteria are always kept in the lab and therefore do not pose any risks to the environment. However, when our project Bio-E evolves into a product, like the using of the system in a biorefinery, there is certainly a chance that they may make their way to the environment. (ex. to the soil, potentially harming soil microbiota) One way we prevented this is by using genes that are totally harmless and actually can counter some of the of toxic chemicals. The gene FucO is a part of the bacteria’s own metabolism to fight harmful substances such as the furans whereas GSH is a part of each and every living tissue, responsible for the scavenging of free radicals and reactive oxygen species. Therefore, even in the unlikely event of our bacteria contacting the environment, our engineered bacteria shouldn’t pose any risks. Moreover, the bacteria we have used, E.coli KO11 and DH5-alpha are strains of bacteria that are specially engineered with all these concerns in mind and are non-pathogens that do not have any detrimental effects on living cells.

Safe Shipment:

Safe and secure shipment is important for preventing part submission delays and blockages. Our genes were totally safe, not containing dangerous genetic material as they were classified as BL-1. We submitted our parts with standard DNA Submission Kit and followed the iGEM DNA Submission protocol. The labeling was visible and clear in addition to the clarification: “non-hazardous, non-regulated, non-infectious, for research purposes only”.

References

• Lonnie O. Ingram, Kazuyoshi Ohta, Brent E. Wood, (1998), Recombinant cells that highly express chromosomally-integrated heterologous genes, US Patent 5,821,093
• Thermo Fisher Scientific, (n.d.), Ethidium Bromide (EtBr) Dye for DNA and RNA Detection. https://www.thermofisher.com/ng/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/etbr.html
• U.S. Public Health Service Guidelines, (2009), Biosafety in Microbiological and Biomedical Laboratories, 5th edition, pages 59-103
• Wang, X., Miller, E. N., Yomano, L. P., Zhang, X., Shanmugam, K. T., & Ingram, L. O. (2011). Increased Furfural Tolerance Due to Overexpression of NADH-Dependent Oxidoreductase FucO in Escherichia coli Strains Engineered for the Production of Ethanol and Lactate. Applied and Environmental Microbiology, 77(15), 5132–5140. http://doi.org/10.1128/AEM.05008-11