The primary goal of our project is to enhance the production of rhamnolipids in P. putida to use it in bioremediation of oil. There are several possible risks associated with our project:
1. Horizontal transfer of genes
The possibility of gene transfer between accidentally released modified strain P. putida and soil bacteria can result in the development of new pathogens which creates another risk. In addition, spread of mutated strain can have adverse effects on species richness and ecosystem functioning. For that reason, we are going to pay attention to the purification step. Since rhamnolipids are secreted in the extracellular environment, cells are separated through filtration on 0.2 micron membrane filter, and the rhamnolipids in the sterile supernatant will be further characterised through HPLC-MS. No concentration step is required at this stage. While this separation strategy is suitable for the proof of concept phase, it is expensive. A more comprehensive and low-cost separation strategy must be developed if the process is scaled-up.
2. Potential hazard of products of expression
There are no potential risks bound to the products of gene expression. Our final product, rhamnolipid molecules, are simple glycolipids, and they are not of toxicological concern. The breakdown products of rhamnolipids also don't exhibit toxicity to the environment. Rhamnolipids' usage mechanism belongs to the non-toxic mode of action and the product itself is not considered as hazardous waste. It is not flammable and the end-use product contains no combustible liquids and is stable at normal and high temperatures. Hence after biosurfactant production in ex-situ bioreactors, it will be safe to utilize them and be introduced to water sources without any serious precautions.
1. General Lab Safety
Nazarbayev University has provided us with a laboratory from June to October of this year. We followed all of the instructions regarding laboratory safety and COVID-19.
2. COVID-19 restrictions
- We followed strict safety measures in order to prevent the spreading of COVID-19
- Team members who were infected or showed symptoms were not allowed to work until they went through quarantine and provided negative PCR test results.
- Our wet-lab team was divided into two groups and they were allowed to work up to 4 people at a time in a lab. This measure was taken to minimize the chance of spreading the virus.
3. BSL-2 Laboratory guidelines
4. Safety Training
- Wet-lab team members received adequate safety training for working in the BSL-2 laboratory.
- Basic BSL-2 safety protocol, including waste separation, sterilization of glassware, and use of personal protection equipment (PPE) was adopted as per the regulatory agency recommendation and University regulation.
Experimental Design Safety
We designed all of our experiments to comply with standards of the BSL-2 laboratory. In addition, we were always under the supervision of our secondary PI, Dr. Obinna Ajunwa, PhD in Electrochemical Microbiology or in the presence of TAs of the Department of Biology. In any case when students have questions on how to handle or dispose of the chemicals, these people are available to help. Moreover, we are advised to ask the Health and Safety department of Nazarbayev University on issues or uncertainties regarding waste management and safety precautions.
We used microorganisms that comply with the standards of Biosafety Laboratory Level 2. P. putida and E. coli are generally safe organisms with BSL-1.All the experiments involving viable P. aeruginosa cells have been carried out in a class 2 biological safety cabinet following adequate training and under close supervision of a senior researcher. Basic BSL-2 safety protocol, including waste separation, sterilization of glassware, and use of personal protection equipment (PPE) was adopted as per the regulatory agency recommendation and University regulation.
We made sure to use safe inserts in our laboratory including nadE, rhlA/B. These inserts were not shown to have any harmful effect.
In our experiments, we used a dual-inducible duet-expression vector, pRGPDuo2 which was provided to us by Dr. Rahul Gauttam, Postdoctoral Research Fellow at LBNL.
We used several hazardous chemicals including Phenol, Chloroform, Ethidium Bromide, and Phenol-Chloroform-Isoamyl-Alcohol. All of the chemicals were stored in special shelves. All of the experiments involving these chemicals were done according to safety procedures by first studying the safety data sheets, and then performing the experiment in a chemical fume hood. All of the work was performed in safety gloves and safety goggles. In case of accidents, there were sink and eyewash stations next to the fume hood. Below you can find safety data sheets that we referred to during the design of the experiments.