Safety is always standing at the top position during our project designing and performing. In a well-organized laboratory, all NEFU-China members have received safety training before starting their experiments. Additionally, we have considered the possible biohazard issues.
According to the World Health Organization Laboratory Biosafety Manual, the rank of our lab is classified as BSL1. Each member was informed of the safety rules and emergency handling. Experiment regulations were strictly observed. During all the experiments, we always wear lab-gowns, rubber gloves and surgical masks. 70％ alcohol was applied to listerize anything that may contact bacteria before and after experiments. In addition, both liquid and solid biohazard waste are autoclaved before disposal.
We have subcloned genes related to the autoinducer-2 response in Salmonella and integrated them into Lactobacillus genome. Then our engineered bacteria can import AI-2 molecules secreted by pathogens in spoiled yogurt and activate the expression of the report gene to produce the blue pigment.
We needed the genes related to the autoinducer-2 response in Salmonella, so we extracted genomic DNA of Salmonella Typhimurium str. LT2 and amplified these essential genes by PCR. We used Escherichia coli MC1601 for molecular cloning. Besides, we cultured Bacillus subtilis H9 and Escherichia coli CD-2 in order to determine the correlations between their growth and AI-2 production.
All the organisms used in our project are in Risk Group 1 except for Salmonella typhimurium, which is in Risk Group 2. Since we just need the genomic DNA of Salmonella for gene cloning, we conducted the DNA extraction in a biosafety cabinet. Thus, we do not have substantial contact with live Salmonella.
Our project aims to create a detecting system that can be potentially used in yogurt fermentation. Our chassis can still be probiotics and benefit intestinal health. The report gene we used is a type of chromoproteins, harmless to both human and the environment. Obviously, genetically engineered Lactobacillus needs extensive test and inspection for its possible use in food fermentation to produce safe or eatable yogurt. To avoid the potential risks that may be raised by genetically engineered food, we think of making small tubelets with our yogurt guarder. Each tubelet can be attached to a cup with regular yogurt fermented at the same condition of the tubelet yogurt. If the testing yogurt in a tubelet turns blue, it will suggest the spoilage of the yogurt in its attached cup (See Future applications for more details). Besides, our future study will focus on food-grade selection makers to replace currently used antibiotic resistance genes. We will also minimize the elements used for genetic engineering.