Before starting the work, we were well trained on the safety of working with microorganisms. As undergraduate biotechnology students, we received a lot of theoretical and practical training on safe laboratory work as part of our lesson plan. All team members gained laboratory experience during various internships and placements. In addition, we received special training from our instructor Marcin, who introduced us to the laboratory where we were conducting the project and provided assistance as we learned new laboratory techniques. Moreover, many of our questions were also being answered by Dorota Krzyzanowska, Ph.D, who is a lab manager.
During our daily work, we were guided by some of the most important rules:
First of all, the Intercollegiate Faculty of Biotechnology UG&MUG in Gdańsk where we conducted the experiments is a modern facility suitable for safe laboratory work with various types of biological material. The unit has the status of a genetic engineering facility confirmed by the Ministry of Climate and Environment, which means that work with genetically modified organisms (GMOs) can be carried out here in a way that limits the danger to humans and the environment. Our experiments were conducted in the biosafety level 2 laboratory, access to which is restricted. In our work we used a lot of safety gear and equipment available in the laboratory. Much of the work with microorganisms was conducted in a Biological Safety Cabinet (BSC) to minimize time spent at the Bunsen burner, which provided greater safety and reduced the risk of burns. The laboratory is equipped with all the equipment needed to work with microorganisms: incubators, -80'C freezers, refrigerators (divided into refrigerators for clean chemical reagents or samples that may be contaminated with bacteria or DNA), chemical fume hoods, ultracentrifuges, thermoblocks, PCR thermocyclers and autoclaves. Waste from the laboratory is separated and disposed of properly: biological material is sealed and sterilized in an autoclave, used chemical reagents are collected in appropriate containers and picked up by an appropriate company.
In the course of our work, we came into contact with many commonly used chemical reagents. However, some of them required our increased attention. Such substances turned out to be antibiotics used in the culturing of microorganisms (ampicillin, kanamycin, chloramphenicol) and chemical compounds: dibutyl phthalate, monobuthyl phthalate, buthyl benzoate, benzoic acid and phthalic acid. In order to reduce the risk to us and the environment we have familiarized ourselves with the with their official safety data scheets (SDS) before working with them. We took special care when working with mixtures that contained them, and placed them in a specially prepared separate waste container.
The design of our project involved minimizing the potentially hazardous effects of work involving biological material. The bacterial strains used are Escherichia coli DH5-alpha and E. coli BL21. Both of these are non-pathogenic strains commonly used in genetic engineering. They are well studied and pose no threat to humans or the environment and are included in the White List. The gene fragments we used for genetic modification were synthesized for us by IDT and TWIST, which are members of IGSC (International Gene Synthesis Consortium). When working with bacteria, our instructor, PI or other lab employee was always nearby. Our mentors were there to help us with transformation troubleshooting and make sure we worked in a safe manner.
Figure 2. Graphic representation of our enzyme complex immobilized on cellulose.
One of the milestones of our development was to decide on a cell-free design for the project. Initially, we planned to immobilize live bacteria that would carry out enzymatic degradation of phthalates, however, we changed our decision when we discussed the actual application of our solution. A water purification filter containing live bacteria cells could not be used in real life environment. We did not want our project to assume the eventual release of GMOs into the environment, as this would have required years of research into the possible effects of such an action. Thus, our final design involves the use of purified enzymes immobilized to cellulose via scaffoldin, a structural protein. In this case, our product contains only proteins, which eliminates the risk of uncontrolled spreading of biological material in the environment, which could happen if bacteria were used. The enzymes we used do not pose a threat to humans or the environment, and could be sold as a finished product in the future, similar to enzymes commonly used in washing powders. You can read more about our decision on the Human Practices Page.
