Introduction
Our team is aware of the strict practices required to work safely with chemicals, microorganisms and GMOs in a laboratory environment. All of our wet lab team members have experience in a laboratory setting and were therefore well-versed with good laboratory practice before our iGEM journey. However, this does not diminish the importance of learning the specific safety protocols from a new laboratory to maintain a safe working environment. Ensuring safety is not only important for our team but also for the well-being of the other people in the lab, as well as the environment outside of the lab. Therefore, we approached our work with utmost care and dedication, as documented on this page.
General Laboratory Safety
We conducted our experiments in two different labs, the first one being the laboratory of our PI, Vitor Pinheiro, which is situated at the Rega Institute for Medical Research on the Gasthuisberg Campus, on the outskirts of Leuven. The Rega Institute's location next to the University Hospital UZ Leuven and the Fire Department of Leuven ensures swift handling in case of an emergency. The Pinheiro lab also has plenty of experience supervising KU Leuven iGEM teams, acquired over many years. The second laboratory is led by Professor Patrick Van Dijck and is located at Campus Arenberg. Professor Van Dijck and his team have extensive experience with rigorous safety precautions when handling fungal species, ranging from industrial production yeast strains to pathogenic fungal species. Here, we handled our Yarrowia lipolytica and Saccharmoyces cerevisiae experimental work.
The laboratory of Dr. Pinheiro adheres to ML-1 lab requirements and follows iGEM safety and security policies. All team members involved in wet lab practices at the Pinheiro lab underwent comprehensive laboratory training focusing on Health, Safety, and Environment (HSE) matters. Thanks to this training, which was supervised by an HSE liaison, team members were legally permitted to work in the laboratory, as required by internal KU Leuven policy.
The laboratory safety training we received consisted of two parts. The first part involved discussing the orientation checklist, covering various HSE aspects including emergency procedures, first aid and Good Laboratory Practice (GLP). To comply with GLP, we wore personal protective equipment (PPE, most importantly gloves, lab coats, and closed shoes) at all times. Additionally, we tied back long hair, as seen in the accompanying picture. We also adhered to standard GLP practice like refraining from eating or drinking in the lab, as well as always washing hands upon entering or leaving the laboratory. We were mindful when using collective protective equipment. An example of this consisted of ensuring proper use of the fume hood without using it for storage, as well as keeping doors closed for appropriate ventilation. We followed specific procedures for the disposal of chemical and biological waste and maintained a clean and orderly lab environment. Moreover, we strictly followed the proper corridors of circulation in the vicinity of the lab space to ensure strict separation between any chemical and biological material and office spaces nearby.
Wet lab member handling bacterial culture work
according to GLP under a flow hood.
She wears appropriate PPE and has her hair tied up.
Our bench at the Pinheiro laboratory.
The practical training in the laboratory involved learning site-specific procedures to adhere to the lab's safety protocols. The training covered the location and usage of fire extinguishers, familiarization with the first aid kit, identification of showers and eye wash stations in case of emergencies, and site-specific disposal procedures for chemical and biological waste. We also learned standard laboratory procedures.
The Van Dijck laboratory also adheres to ML-1 lab requirements and similar safety protocols to those described above for the Pinheiro laboratory were handled. To separate our strains from the other microorganisms in the lab, we worked on a separate floor and had a dedicated biosafety cabinet for the iGEM team. Moreover, our team also received the proper safety training for the ML-2 space in the building, albeit all our work was carried in the ML-1 area.
To complement our trainings, we attended the biosafety workshop by iGEM IISER-Pune-India on the 17th of July. We discussed biosafety, and how to take those aspects into consideration when doing synthetic biology research.
Our fire extinguisher and first aid kit at the Pinheiro laboratory.
Our emergency shower at the Pinheiro laboratory.
Biosafety cabinet at the Van Dijck laboratory.
Project-Specific Safety
Microbial Strains
Vectors
For our experimental work in E. coli, we worked with pET-29 plasmids. The pET vector system for recombinant protein expression in E. coli employs the T7 transcription and translation regulatory system. It features a T7 promoter and a lac operator (LacI) sequence downstream, controlling gene expression. By adding a lactose analog, the inhibitory action of LacI is blocked, inducing T7 RNA polymerase expression and enabling high-level protein production. Our plasmid also contains a kanamycin resistance gene, a linker sequence and a superfolding variant of GFP (sfGFP).
Our expression vector in S. cerevisiae was a pBEVY plasmid, used for recombinant protein production in our strain of choice, Ethanol Red. Our proposed cloning strategy in S. cerevisiae relies on the pBEVY plasmid containing a β-Lactamase sequence expressed under a bacterial promoter, to allow for ampicillin-based selection of E. coli cells carrying the assembled constructs of interest. For selection in the yeast transformation, our constructs include an expression cassette coding for Nourseothricin acetyltransferase, conferring transformed yeast strains resistance to nourseothricin.
Our proposed genetic engineering strategy in Yarrowia lipolytica used an NDV-URA3 vector backbone, with an ampicillin resistance cassette for selection in producer E. coli strains, as well as an URA3 cassette for auxotrophy-based selection in the yeast strain.
Inserts
The genetic constructs that we used consisted sfGFP linked with our biosurfactant or hydrophobin insert of choice. Biosurfactants are used for various industrial applications, therefore it is unlikely that our recombinant biosurfactant protein is toxic4. As hydrophobin proteins have great importance in the food industry and are sometimes biproducts of industrial processes such as beer production, there are no major concerns around the safety of our recombinant protein products 5. In S. cerevisiae, the cloned sequences were placed under the expression regulation of an ADH1 promoter. A secretion signal derived from the endogenous Mating factor alpha-1 sequence was included as an N-terminal fusion with our proteins of interest.
Chemicals
We used SYBR-Safe (mutagen) to visualize the DNA bands in agarose gels and acrylamide (neurotoxin) for SDS-PAGE gels. We also used a phenol-chloroform mixture (irritant, corrosive) to extract yeast DNA. For these chemicals (SYBR-Safe, acrylamide and the phenol-chloroform mixture), we followed the proper standards of practice (SOP) while exclusively working under a chemical hood. We used NaOH solution to adjust pH of yeast media. For DNA purification, we made use of chaotropic salts as provided in our purification kits. For surface sterilization we used 96% and 70% ethanol. To sanitize liquid culture waste, we used Virkon (skin irritant, serious eye damage) Being aware of the safety hazards, we wore appropriate PPE during the execution of experiments with these chemicals.
- Potts, C. Statement of nonpathogenicity. Thermofisher (2016).
- NIH guidelines for research involving recombinant or synthetic nucleid acid molecules (2016, April). National Institute of Health.
- Jach, M. E. & Malm, A. Yarrowia lipolytica as an Alternative and Valuable Source of Nutritional and Bioactive Compounds for Humans. Molecules 27, 2300 (2022).
- Ingsel, T., De Souza, F. M. & Gupta, R. K. Biosurfactants for industrial applications. in Green Sustainable Process for Chemical and Environmental Engineering and Science 467–493 (Elsevier, 2022).
- Khalesi, M., Gebruers, K. & Derdelinckx, G. Recent advances in fungal hydrophobin towards using in industry. The Protein Journal 34, 243–255 (2015).