Team:CGU Taiwan/Safety
Safety
1.Brief introduction of the project
Transgenic yeast is designed to be activated by light, being able to secrete out the enzyme which mainly catalyze cellulolysis. Instead of using chemicals, we introduced a new model to detach the ink particle from paper fiber specifically.
2.The organisms we used
east (Saccharomyces cerevisiae), E. coli (DH5α)
3.The parts we used
(1) Three enzymes for cellulolysis endo-beta-1,4-glucanase B, endo-1,4-beta-xylanase A and lipase ORF sequence are from the genome of Aspergillus oryzae, Neocallimastix patriciarum and Rhizopus niveus respectively. (2) Ste12 promoter and protein of positive up-regulation We retrieved the sequence of ste12 promoter from upstream 500bp of a pheromone-related pathway transcription factor of Saccharomyces cerevisiae. And we directly acquired the sequence of ste12 protein which is the product of this pathway. (3) Improved GAL4 based yeast light-switchable promoter system We adapted the sequence of GAL4 based yeast light-switchable promoter system from BBa_K801042, which is originally designed by Dong-Jiunn Jeffery TRUONG and the group iGEM12_TU_Munich.
4.The operation risks during experiments and their solution
(1) Molecular cloning of E. coli In order to make DNA visible on agarose gels, ethidium bromide was used for staining. It could insert itself between double stranded DNA of samples as well as ours and act as a mutagen which affects DNA biological processes, such as DNA replication and transcription. To prevent skin contact, we not only used protective gloves but also changed it frequently to avoid any ethidium bromide left over. All materials that contact with ethidium bromide were considered polluted and then they should be separately disposed of. (2) Molecular cloning of yeast To select the transgenic yeast we interested, the success one will grow up with drug resistance. As we have selected out the yeast wanted, the remaining medium is into the discard, but there is still great amount of yeast in it. If transgenic yeast which has drug resistance flows out, it might become booming and harmful in the nature. To deter from this happening, we added bleach into the medium or sent to sterilization for security. (3) Test of enzyme activity We detected the amount of total carbonhydrate with concentrated sulfuric acid and phenol sulfuric acid. They’re both strong reagent which may cause serious dehydration toward our skin. Besides the lab coat and protective gloves, we even wore goggles to prevent from any possible contact.
5.Any safety issues raised from the devices?
Environmental issue As the device will spray the transgenic yeast onto a paper, there might be a question whether it’s going to stay alive or not after the reprocess. The answer is negative because one of the process will heat to high temperature which kills the yeast for sure. Second, since our device isn’t sealed up completely, the yeast coming out from spray-head will be hard to be removed from the border of box and it might contaminate the gene pool in the environment if they’re not sterilized from the abandon boxes. Thus, we should treat the device a disinfectant to stay clean and safe after use.
