Safety and Security
The Laboratory of Genetic Engineering and Molecular Diagnosis was the designated laboratory to be used during the design and development of our project. It is located at the FEMSA Biotechnology Center inside our university, which is where most academic researchers carry out their work. This specific laboratory has limited access controlled by fingerprint technology in which only certain professors and members of authorized research groups are allowed to access.
Regarding safety measures, our lab is equipped with an eye wash fountain, a safety shower, two laminar flow cabinets with UV sterilization, an autoclave, a delimited space for bio-hazardous waste and broken glass material. In addition, the lab contains information about the correct classification of waste according to its hazard level, all of the contaminated materials that are generated in the lab are autoclaved and then handled by an authorized infectious biological management third-party service for proper disposal. For every day work we use a level 2 biosafety cabinet. [1] In the same BSL-2 lab. It is important to notice that our institution provides biosafety and security for everyone working in the lab and we are also under constant supervision of the administration office that ensures our safety.
On February 2022 our team leaders took a safety course by the FEMSA Biotechnology Center. However, all of our team members who are working on the lab took a course on biosafety imparted by IGEM ambassador Emiliano Gonzalez, the past June 22nd , 2022.
Project safety
Our construct designs aim to provide the M13 phage the capacity to infect Pseudomonas by modifying the P3 gene, changing the domain that allows the specific interaction with the bacteria, a technique that is widely used and known as phage display. This synthetic interaction will be used to deliver the sRNA's to silence the antibiotic resistance genes, however, it is important to note that we did not reach this stage of the project since we are having difficulty with previous parts of the project. The whole system will be tried first in E. coli, as a proof of concept and we will move on to Pseudomonas. We are proposing a delivery system (only in theory) based on nebulizing nano encapsulated bacteriophages in liposomes, in order to ensure both effectiveness and aid to its safety.
| Chasis |
Description |
Risk Group |
Possible risks |
| Escherichia coli (DH5-ɑ & BL21) |
Escherichia coli is a gram-negative bacterium commonly used in
research laboratories, due to its rapid growth rate, simple nutritional requirements, easy
transformation, well-established genetics and genomic sequence.
|
Group 1 |
Risk group 1 organisms do not cause any disease in humans. Lab coat and gloves are needed. |
The ability of our parts effectiveness and its delivery through phages assembled by E. coli TG1 were tested in E. coli DH5 ,BL21, and DH10.
Biosafety of other components of our project
| Components |
Description |
Risk Group |
| Pseudomonas aeruginosa (PAO1) |
Pseudomonas aeruginosa is a common, free-living, Gram-negative bacterium that can cause significant disease as an opportunistic pathogen. It´s a common model organism to study Gram-negative opportunistic pathogens and basic microbiology.” [2]
|
Group 2 risk organisms can cause moderate individual risk, and exhibit low community risk) - These pathogens can cause human or animal disease but are unlikely to be a serious hazard to lab workers, the community, livestock or the environment. [3]
|
| Bacteriophage M13 (PAO1) |
“Bacteriophages are bacterial viruses and consist of a single- or double-stranded DNA or RNA protected by a protein capsid.” [4]
|
iGEM´s white list of bacteriophages
|
As part of the safety guidelines for our project, we registered a whole organism to the Check-in. We registered Pseudomonas aeruginosa (PAO1) as it was not in iGEM´s white list and it belongs to risk group 2. Pseudomonas aeruginosa (PAO1) is an opportunistic pathogen. [2] Because of this, exposure to this microorganism by someone with a compromised immune system could be potentially dangerous, as well Pseudomonas is very likely to develop antibiotic resistance (which is will be necessary for our extended proof of concept). We have not used Pseudomonas yet, as we only evaluated the effectiveness of our project in Escherichia coli ; nonetheless , the use of Pseudomonas might be employed at the following stages of our project to prove that our M13 phage display was successful. We have taken the necessary precautions , and decided that we will not be performing such tests , and in case of performing them , it will be our Ph.D. advisors who perform the trials in a BSL2 pathogen laboratory that is suitable for handling these types of microorganisms.
On the other hand, General Biosafety Recommendations for the Contained Use of Bacteriophages described by [4] were followed for the infection of E.coli TG1 using M13 bacteriophages . These recommendations include:
- Respecting good microbiological practices.
- Using appropriate disinfectant prior to autoclaving non-disposable equipment.
- Cleaning working surfaces regularly with proper disinfecting solutions.
- Cleaning up any spill immediately and disinfecting correctly.
- Avoid procedures generating aerosols.
- Not opening flasks during cultivation unless absolutely necessary.
- Avoiding moisture by drying all glass and plastic materials.
Other guidelines we decided to follow for the infection protocol we performed:
- Using a separate laminar flow cabinet to avoid contaminating other people's work.
- Contact the biosafety committee in our institution to ask for permission to manipulate bacteriophages.
References
- LaBauve, A. E., & Wargo, M. J. (2012). Growth and Laboratory Maintenance of Pseudomonas aeruginosa. Current Protocols in Microbiology, 25(1). https://doi.org/10.1002/9780471729259.mc06e01s25
https://doi.org/10.1002/9780471729259.mc06e01s25
- Basics of Biosafety Level 2 | Office of Clinical and Research Safety. (2022). Vumc.org. https://www.vumc.org/safety/bio/basics-biosafety-level-2#:~:text=Basics%20of%20Biosafety%20
Level%202%201%20Limit%20access,%28e.g.%2C%20safety%20devices%20or%20non-sharps%29%20exist.%20M%C3%A1s%20elementos
-
United States Department of Health and Human Services (2015). Risk Groups. https://www.phe.gov/s3/BioriskManagement/biosafety/Pages/Risk-Groups.aspx#:~:text=WHO%20Risk%20Group%202%20
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Verheust, C., Pauwels, K., Mahillon, J., Helinski, D. R., & Herman, P. (2010). Contained use of bacteriophages: risk assessment and biosafety recommendations. Applied biosafety, 15(1), 32-44.