Because our main goal consists in the development of a platform that allows us to find new antibiotics for treatment against Pseudomonas aeruginosa we designed the following working plan concerning whole cell biosensors and a mucolytic.
Lab safety
Our Laboratory of Genetic Engineering and Molecular Diagnosis is located in the FEMSA Biotechnology Center of our university were most of scientific research and academic laboratories are. Not everyone can come into this laboratory; there is limited entry and only by a fingerprint technology access professors and members of authorized research groups are allowed in specific work schedules.
As to safety concerns, our lab is provided with an eye wash fountain, a safety shower, an autoclave, two laminar flow cabinets with UV sterilization and a special delimited section for bio-hazardous waste and broken glass material. Our lab is equipped with special containers and instructions for the correct classification of waste according to its level of danger.
In the designing of our project and protocols we made sure to avoid any toxic materials and compounds that could be harmful to us and the environment. That is why we looked for safer materials that could be a substitute to dangerous ones. For example in an electrophoresis gel instead of using ethidium bromide, which is mutagenic and carcinogen, we opted for much safer option: the SmartGlow Stain that works the same. Also in the choosing of the organism in which the sialidase protein is naturally expressed we make sure it was a non-pathogenic one. In this case we chose the protein sequence in M. Viridifaciens, a bacteria found in human’s mouth microbiota making it a non-hazardous enzyme that when expressed and purified represented minimum risk.
Strains
In our project we used E. Coli and Bacillus subtilis strains which are non-pathogenic, this means that the risks of using these organisms is very low for the environment and laboratory staff.
| Chasis | Description | Biosafety Level | Possible risks |
|---|---|---|---|
| Escherichia coli (DH5-ɑ) | 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. | Level 1 | Long skin exposure can cause irritation, dryness and redness in the skin. It is only toxic if inhaled in large quantities. |
| Bacillus subtilis | Bacillus subtilis is a typical gram-positive eubacterium that is known to be spore-forming and has been used particularly to study that process in prokaryotes. | Level 1 | If contact with the skin it may cause irritation and is only toxic if inhaled in large quantities. It could be considered as a non-risk product. |
Safety protocols
Our experiments were checked and approved by our advisors; the protocols were based on other iGEM teams protocols, papers, company kits and recommendations. We followed up the already established rules to control the work in the laboratory and reduce the risks involved in manipulating biological material. Our priority was having appropriate working practices, technical measures and the use of personal protective equipment: nitrile gloves and suitable work clothes (lab coat, long pants, long-sleeved shirts, socks and closed shoes). During the time we carry out our experiments we didn’t face any major risks as we were very precocious and followed every safety measure indicated:
| Protocol | Hazardous Materials | Safety measures | Type of hazard |
|---|---|---|---|
| Characterization of Amplification cascade with Heavy metals | Mercury chloride and cadmium chloride | Fatal at contact with skin; causes severe skin burns and eye damage and suspected of causing genetic defects. Very toxic to aquatic life with long lasting effects. As we didn’t get to work in the characterization we didn’t managed this compound. But only store it in a safe cabinet, in this we make sure to do it in a ventilated place, use nitrile gloves-lab coat and disposed the contaminated material in the proper waste container. |
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| Characterization of Amplification cascade with Heavy metals | Mercury chloride and cadmium chloride | Fatal at contact with skin; causes severe skin burns and eye damage and suspected of causing genetic defects. Very toxic to aquatic life with long lasting effects. As we didn’t get to work in the characterization we didn’t managed this compound. But only store it in a safe cabinet, in this we make sure to do it in a ventilated place, use nitrile gloves-lab coat and disposed the contaminated material in the proper waste container. |
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| SDS-Page |
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For a SDS- page a polyacrylamide gel is done by adding TEMED and PSA to Acrylamide, which are harmful for the respiratory system so we were extremely precautious to don’t inhale and work in a ventilated place. Also acrylamide is known for being a strong neurotoxin. ß-mercaptoethanol and SDS have toxic effects, especially ß-mercaptoethanol so it was necessary to minimize the time of exposure. To prevent any risk we used nitrile gloves and worked in within the designated space. |
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| Protein purification columns | Imidazol | Imidazole works as a detergent and can be very harmful to the skin so it is a must to wear gloves at all times. Eye protection is recommended as well, but it is not essential. In case of contact, the surface must be thoroughly washed. |
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| Protein purification columns | Nickel sulphate | May cause an allergic skin reaction, skin irritation and is suspected of causing genetic defects and cancer. To prevent any damage we used personal protection like safety eyeglasses, nitrile gloves and lab coat. |
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Ethics
Our team took very seriously the ethical aspect and responsibility that concerns a synthetic biological project. Thus, every team member signed a letter committing to follow the safety rules inside the lab, wear proper protective clothes, report any issue with any equipment, among others. Coupled to this, every member of the team that worked in the wet-lab received training by Berenice Enríquez, the coordinator of FEMSA Biotechnology Center, to know better the safety rules in the laboratory and prevent any accident. Also we received coaching workshops from Carolina Elizondo, one of our advisors, on many lab protocols to gain experience and learn how to correctly use the lab equipment. Thus, at all times we listened to our advirsor’s suggestions on experiments and protocols, they helped us be aware of all possible risks and change paths if necessary. We were also in contact with biological reagent suppliers for any security questions that arose throughout the project.