Our lab at TAS is classified as Biosafety Level 1, which means the lab uses microbes that pose little to no risk to healthy individuals. Hazardous chemicals and solutions are used at a minimal level and in well ventilated areas such as fume hoods in the TAS research lab. For instance, we do not use EtBr for gel electrophoresis. Instead, we use a safer nucleic acid stain called Seeing Safe DNA Dye. Nevertheless, all chemicals and solutions are still treated with all due respect, care, and caution. MSDS are stored on our lab computers and in a folder on the lab technician's desk.
For the vast majority of experiments, we work with a safe and common lab strain, Escherichia coli (E. coli) K-12 DH5alpha, and follow safety rules set by our lab instructor. We have our own biosafety committee, which consists of three research teachers Dr. Teresa Chiang, Mr. Jude Clapper, and Mr. Alex Dezieck. They oversee proper work area conditions by checking on disposal of Petri dishes and liquid wastes, sanitation, and teaching proper laboratory techniques. Our guidelines, taken from National Yang-Ming University's Center of Environmental Protection and Safety, cover many safety rules and procedures ranging from lab specific rules to behavior. For example, we prohibit food, open-toed shoes and drinks in the lab. We also have a thorough clean up procedure. We also have a thorough clean up procedure. For example, all bacterial liquid wastes are bleached and used tips are autoclaved before disposal. Other lab wastes are carefully packaged and then sent to a disposal facility to ensure no hazardous materials are mismanaged.
In Taiwan, there are many laws and regulations regarding biosafety in research labs. These can be found in the National Yang-Ming University's Center of Environmental Protection and Safety and Health page linked here. Guidelines can be found here.
PROJECT-SPECIFIC BIOSAFETY
Acetaldehyde
Acetaldehyde is an intermediate in the conversion of alcohol to acetate. In order to test the functions of ALDH2*1 and ALDH2*2, we needed to use acetaldehyde as the substrate. Acetaldehyde is a highly flammable liquid and considered a possible human carcinogen (Group 2B) (International Agency for Research on Cancer, n.d.; Click here to download the MSDS from Sigma Aldrich). The acetaldehyde used in our experiments was synthesized by our advisor, Mr. Clapper. To ensure safety, experiments were performed inside a variable air volume chemical fume hood, with lab gloves, lab coat, and eyewear for eye protection. All waste products were sealed in waste bins, picked up by a local waste removal company (THEPS Limited) and transported to Cheng Gong University for disposal.
Bacteria Strains
We work mainly with E. coli K-12 DH5a, one of the most common lab strains (Environmental Protection Agency, 1977). For our project this year, we have also chosen to deliver recombinant ALDH2 using probiotic strains. We first tried Lactobacillus casei (L. casei), a common strain found in food products such as yogurt (Yakult, n.d.), but we failed to transform L. casei. Next, we tried two commercially used and nonpathogenic probiotic strains: L. lactis and E. coli Nissle 1917. L. lactis is used in the production of dairy products such as buttermilk and cheese, while E. coli Nissle 1917 is mainly used for the treatment of gastrointestinal disorders (Leroy & De Vuyst, 2004; Grozdanov et al., 2004; Hancock et al., 2006; Secher et al., 2018; Mutaflor, n.d.a). Several research groups have genetically engineered E. coli Nissle 1917 and L. lactis to deliver proteins for disease treatment, and these are currently in clinical trials (Braat et al., 2006; Bahey-El-Din et al, 2010; Synlogic, n.d.).
All mentioned bacteria strains are classified as Risk Group 1 (DMS-8741, n.d.; DMS-4366, n.d.; Mutaflor, n.d.b), and if bacteria were to escape the lab they would pose low risk to the community. After use, all bacterial wastes were either autoclaved or bleached to kill the microbes before disposal.
REFERENCES
Bahey-El-Din M & Gahan CG. (2010). Lactococcus lactis: From the dairy industry to antigen and therapeutic protein delivery. Discov Med. 9(48):455-61.
Braat H, Rottiers P, Hommes DW, Huyghebaert N, Remaut E, Remon JP, van Deventer SJ, Neirynck S, Peppelenbosch MP, Steidler L. (2006). A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn's disease. Clin Gastroenterol Hepatol. 4(6):754-9.
DSMZ. (n.d.). DMS-8741. Retrieved from https://www.dsmz.de/catalogues/details/culture/DSM-8741.html?tx_dsmzresources_pi5%5BreturnPid%5D=304.
DSMZ. (n.d.). DMS-4366. Retrieved from https://www.dsmz.de/catalogues/details/culture/DSM-4366.html?tx_dsmzresources_pi5%5BreturnPid%5D=304.
Environmental Protection Agency. (1977). Final Risk Assessment of Escherichia Coli K-12 Derivatives. EPA. Retrieved from https://www.epa.gov/sites/production/files/2015-09/documents/fra004.pdf.
Grozdanov L, Raasch C, Schulze J, Sonnenborn U, Gottschalk G, Hacker J, Dobrindt U. (2004) Analysis of the genome structure of the nonpathogenic probiotic Escherichia coli strain Nissle 1917. J Bacteriol. 186(16):5432-41.
Hancock V, Dahl M, & Klemm P. (2010). Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation. J Med Microbiol. 59(4):392-9.
International Agency for Research on Cancer. (n.d.). List of Classifications, Volumes 1-122. Retrieved from https://monographs.iarc.fr/list-of-classifications-volumes/.
Kimoto H, Kurisaki J, Tsuji NM, Ohmomo S, Okamoto T. (1999). Lactococci as probiotic strains: adhesion to human enterocyte-like Caco-2 cells and tolerance to low pH and bile. Lett Appl Microbiol 29(5):313-6.
Leroy F & De Vuyst L. (2004). Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol. 15(2): 67-78.
Mutaflor. (n.d.a). About Mutaflor. Retrieved from https://www.mutaflor.com.au/about-mutaflor/.
Mutaflor. (n.d.b). Medicinal product safety. Retrieved from https://www.mutaflor.com/mutaflor-with-escherichia-coli-strain-nissle-1917-for-human-gut-health/medicinal-product-safety.html.
Secher T, Maillet I, Mackowiak C, Le Bérichel J, Philippeau A, Panek C, Boury M, Oswald E, Saoudi A, Erard F, Le Bert M, Quesniaux V, Couturier-Maillard A, Ryffel B, (2018). The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep. 8(1):11245.
Synlogic. (n.d.). Creating a synthetic biotic medicine. Retrieved from https://www.synlogictx.com/synthetic-biotics/creating-a-synthetic-biotic/.
Yakult. (n.d.). What is Yakult. Retrieved from https://www.yakult.co.uk/.
