Team:NUDT CHINA/Safety
Safety
This year, we demonstrate a modulization design—LiPrePro to optimize Predator Pro, a modularized protein degradation system we have been working on in the past few years to allow light inducible control of cell cycle. In detail, we would try to achieve light-mediated degradation of endogenous cyclin E, a key regulator protein in cell cycle, thereby manipulating cell cycle in mammalian cells. To introduce light inducible protein degradation in Predator system, we would replace the previous constitutive interaction module with light inducible protein dimerization pairs like CRY2/CIB1, LOV2/Zdk1, and PixD/PixE. The Cyclin targeting would be achieved by different targeting modules including cyclin E ScFv, alpha-C Helix of CDK4 etc. Putting safety first, we considered the safety issues as follows:
1. E.coli was used to performing molecular cloning. We choose genetically modified E.coli, which has not acquired any characteristics that enable them to produce hazardous substance or invade the immune system/other systems of human or animals, as our chassis organism in our project. They are harmless from both a personal and public healthy point of view.
2. 293T cells were used to demonstrate the feasibility of the newly constructed plasmid and the Predator Pro. 293T cells, which derive from 293cells, are transfected with gene E1A of adenovirus. Though adenovirus could lead diseases to people, the 293T cells are completely safe.
3. All genes cloned into pcDNA3.1 are non-conjugative, preventing horizontal transfer of our parts. We use CMV and EF-1a as promoter, which guarantees that our bacteria won’t produce large amounts of protein if not induce the expression.
4. All our parts we used are guaranteed to be safe for human and won’t cause any biological pollution to the environment.
1. All our operations comply with the safety regulations of laboratory safety manual.
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. Before we started the experiments, our advisors gave us a wonderful lesson about Lab safety, and the original members provided detailed and comprehensive guidance to new members provided detailed and comprehensive guidance to new members to avoid mistakes in the operation. Here is the emphasis on the specification:
a. Wear white coats, masks and rubber gloves.
b. Get familiar with the functions of each equipment in the lab and can use them properly.
c. Standardize experimental operation and avoid wrong operation.
d. Have a discussion on the procedures of the experiment we will perform to get familiar with the operation steps and clarify all the details.
e. Make the equipment and the appliance prepared before we begin our experiment to avoid delaying the process of the experiment or searching for laboratory appliance when operating.
f. Reasonable and scientific treatment of experimental waste is needed to prevent pollution.
g. Calm down and get ready to deal with possible accidents when operating.
h. Separate members into small groups and groups take responsibility for different experiment steps to raise efficiency.
4. 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.
5. 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.
6. In order to minimize the damage caused to us by the experiment, we used a blue imager to replace the UV imager and the UV glue cutter. Also, we used non-polluting nucleic acid dyes when doing the preparation of agarose gel and agarose gel electrophoresis.