Team:CCU Taiwan/Safety
Team:CCU Taiwan/Implementation
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Biosafety
General Lab Safety roles
• Never work alone in the laboratory without permission and prior knowledge of the instructor.
• Do not engage in rowdy, playful, or unprofessional activities in the laboratory. This includes not being disrespectful of your instructor or classmates.
• Students should wash hands thoroughly after first entering the lab.
• Students should never ever eat or drink anything in the laboratory without explicit permission from the instructor.
• Wear appropriate clothing at all times in the laboratory.
• Wear closed-toe shoes that cover the top of the foot, unless permission otherwise is given by the instructor.
• Wear examination gloves and safety glasses when dissecting or handling cadavers, caustic chemicals, bacterial broth cultures, or as otherwise advised by your instructor.
• Wear gloves when handling any microorganisms. Wear lab aprons or lab coats as advised by your instructor.
• Keep hands away from your face, eyes, and mouth when working with cadavers, chemicals, preserved specimens, microorganisms, or body fluids. This includes not applying cosmetics, not adjusting contact lenses, and not biting your fingernails.
• If any chemicals or other agents splash into your eyes, immediately go to the nearest sink and flush your eyes with water.
• Report ANY and ALL accidents, spills, BREAKAGES, or injuries to the instructor, no matter how trivial they appear.
• Scalpels and other sharp objects can be used only if authorized by the instructor and only after given proper handling instructions. Use small trays to carry all sharp objects. When handling sharp objects, point their tips down and away from other people.
• While wearing examination gloves, students must not leave the laboratory and must not touch any equipment such as microscopes, any personal items such as cell phones, or any door knobs.
• Do not use any lab equipment without instruction and authorization from the instructor. Report any damaged or broken equipment to your instructor immediately.
• Lab benches should be kept free of extraneous items while conducting experiments. This includes unnecessary books, backpacks, cell phones, and other personal items.
Lab safety roles of E. coli operation
The E. coli strains DH5α and BL21 applied in our experiment for cloning and protein expression belong to the WHO-defined Risk Group 1, which do not cause serious human or animal diseases.
1. Operating bacterium related experiments in the laminar flow cabinet.
2. All biowaste would be sterilized by steam sterilization.
3. Report all accidents, spills, breakages, or injuries to the instructor.
4. Use small trays to carry all sharp objects. When handling, point their tips down and away from other people.
5. While wearing examination gloves, must not leave the laboratory and do not touch any equipment, personal items or door knobs.
6. Report any damaged or broken equipment to your instructor immediately.
7. Lab benches should be kept free of extraneous items while conducting experiments.
Lab safety roles of S. aureus operation
The S. aureus strain ATCC 6538, which is applied in antimicrobial function tests, is classified as biosafety level 2 (BFII) in Taiwan. We cultured S. aureus and conducted experiments in the BFII laboratory following the biosafety rules from Taiwan CDC .
1. Conducting S. aureus in biosafety level 2 lab.
2. Don’t release the bacteria out of the lab.
3. Operating bacterium related experiments in the laminar flow cabinet.
4. All biowaste would be sterilized by steam sterilization.
5. Report all accidents, spills, breakages, or injuries to the instructor.
6. Use small trays to carry all sharp objects. When handling, point their tips down and away from other people.
7. While wearing examination gloves, must not leave the laboratory and do not touch any equipment, personal items or door knobs.
8. Report any damaged or broken equipment to your instructor immediately.
9. Lab benches should be kept free of extraneous items while conducting experiments.
Personal safety
Personal safety roles for operating E. coli
1. Wearing latex gloves when operating experiments.
2. Do not engage in rowdy, playful, or unprofessional activities in the laboratory.
3. Wash hands thoroughly after first entering the lab.
4. Do not eat or drink anything in the laboratory.
5. Wear closed-toe shoes that cover the top of the foot
6. Keep hands away from your face, eyes, and mouth.
7. Not applying cosmetics, adjusting contact lenses, and biting your fingernails.
8. Lab benches should be kept free of extraneous items while conducting experiments.
9. If any agents splash into your eyes, immediately flush your eyes with water.
Personal safety roles for operating S. aureus
1. Wearing latex gloves when operating experiments.
2. Do not engage in rowdy, playful, or unprofessional activities in the laboratory.
3. Wash hands thoroughly after first entering the lab.
4. Do not eat or drink anything in the laboratory.
5. Wear closed-toe shoes that cover the top of the foot
6. Keep hands away from your face, eyes, and mouth.
7. Not applying cosmetics, adjusting contact lenses, and biting your fingernails.
8. Lab benches should be kept free of extraneous items while conducting experiments.
9. If any agents splash into your eyes, immediately flush your eyes with water.
Project safety
Environmental safety
The AMPs, collagens, chitosan, and alginate are bio-degradable and won’t interfere with the environment.
Application safety
Human cells
AMPs: The antimicrobial peptides we used are DPK-060, Buforin ll-C and D2A21.
The positively charged AMPs can bind on negatively charged membranes to exert their functions. The external and internal bacterial membranes are both negatively charged, while the eukaryotic cell membrane is only negatively charged on the internal surface. Therefore, AMPs are less likely to bind on eukaryotic cell membranes. Moreover, cholesterol in the eukaryotic cell membrane can also prevent AMP from disrupting the membrane. To sum up, the charge and membrane composition differences between eukaryotic and prokaryotic cell membranes allow the AMPs to selectively target the bacterial cell membrane [1].
CPP: The cell-penetrating peptide we used is TAT48-57.
TAT48-57 will not lead to severe cytotoxic effects at concentrations below 10 μM [2].
Cellular organelles
AMPs: The antimicrobial peptides we used are DPK-060, Buforin ll-C and D2A21.
The AMPs used in this project are DPK-060, Buforin ll-C, and D2A21. Only DPK-060 was selected as a cell-penetrating AMP due to its insensitivity to lysosomal endopeptidase Cathepsin S. We consider that DPK-060 is unable to pass through the cell membranes so that it will not escape the phagolysosome. Consequently, DPK-060 will not escape the phagolysosome to harm other organelles. Further examination is necessary to exclude this possibility.
CPP: The cell-penetrating peptide we used is TAT48-57.
According to the results reported by Duchardt et. al., we believe that TAT48-57 will not escape the endosome at concentrations below 5 μM so that they will not harm the organelles [3].
References:
1. Ebenhan, T., Gheysens, O., Kruger, H. G., Zeevaart, J. R., & Sathekge, M. M. (2014). Antimicrobial peptides: their role as infection-selective tracers for molecular imaging. BioMed research international, 2014, 867381.
2. Cardozo, A. K., Buchillier, V., Mathieu, M., Chen, J., Ortis, F., Ladrière, L., Allaman-Pillet, N., Poirot, O., Kellenberger, S., Beckmann, J. S., Eizirik, D. L., Bonny, C., & Maurer, F. (2007). Cell-permeable peptides induce dose- and length-dependent cytotoxic effects. Biochimica et biophysica acta, 1768(9), 2222–2234.
3. Duchardt, F., Fotin-Mleczek, M., Schwarz, H., Fischer, R., & Brock, R. (2007). A comprehensive model for the cellular uptake of cationic cell-penetrating peptides. Traffic (Copenhagen, Denmark), 8(7), 848–866.
