In our project, we'll use engineered E.coli to produce the fusion protein of dCas9 and split-HRP fragments. Those protein will be purified to form an in vitro detection system of miRNA together with other key fragments such as phi29 DNA polymerase. Target miRNA input will be transformed and amplified specifically into a long single-strand DNA through a Rolling Cycle Amplification process using a specific probe and with the existence of phi29 polymerase. dCas9 protein fused with the previously mentioned split-reporting system will be guided to bind with the DNA strand to reach the further amplification and visualization of the previous signal.
1、We choose E.coli as our chassis organism to be genetically modified in our project. Our chassis bacteria have not acquired any characteristics that would enable them to compromise human immune system/other systems or evade detection and destruction by the former or facilitate spread between people/animals, which makes them harmless from both a personal and public health point of view.
2、Our parts are all safe for human, we ensure that none of the genes or parts that we were assembling would act as virulence factors, and that no known pathogens would be involved in our research.
3、All genes were cloned into either pSB1C3 or pET28a, both of which are non-conjugative, preventing horizontal transfer of our parts. We use LacI as promoter, which guarantees that our bacteria won’t produce large amounts of protein if not induce the expression.
Safe lab work
1、Before we start our experiments, our advisors gave us a wonderful lesson about Lab safety. From that, we have learned a complete understanding of experimental risks associated with synthetic biology.
2、We strictly follow the guideline of the WHO lab biosafety manual and the instruction of our instructors. We are required to understand the experimental principle and method completely before the operation.
3、As for electrical safety, the electrical engineer of the lab showed us the circuit layout and told us details about the use of these equipment. When finish the whole experiment that day, we take turns to check these electric appliances. Before using every kind of equipment, we read the instructions over and over again to avoid any safety problems, and we cut off the power immediately when do not use them. Particularly for using centrifugal machine, it is significant to make sure that you have already balanced it and covered the lid well before centrifugation.
4、All wastes are sterilized with proper treatment and finally send to qualified companies. The abandoned bacteria can only be thrown away after high temperature and high pressure sterilization.
5、We are enforced to wear gloves, masks and lab coat once we enter our lab. It is prohibited to bring anything in the lab out including gloves, masks and lab coat. Therefore each time we get out of the lab, we have to take off lab coat, gloves and wash hands with hand sanitizer.
6、We use human serum in the final stage of our project, which may raise some healthy risks. To reduce those risks, we perform all the operation with human serum in another bio-safety level II lab.
7、Inevitably, we are exposed to toxic chemical reagents, such as GelRed, DEPC and so on, the possible expose to UV light may also raise safety concerns. To reduce such risks, separated area were defined and extra protection were performed when operation toxic chemical reagents and UV lights. We put the toxic chemical reagents in the fume cupboard, any relevant operations are all done in the fume cupboard, and we separate a special area for the preparation of agarose gel and agarose gel electrophoresis, we need to wear two pair of gloves to touch anything in this area.
As mentioned above, our DNA parts are absolutely safe. The DNA parts are safely dried down within 96 well plates, sealed completely, and covered with a protective lid as Parts registry requires.