As members of the synthetic biology community, we are to shoulder the responsibilities to conduct experiments safely and securely, and the premise of doing this is to understand what it means to be safe and secure. In iGEM, safety, or biosafety, covers the procedures, practices or other measures used to manage risks from accidental exposure or release. They protect people from bad bugs, while security, or biosecurity, on the other hand, covers the procedures, practices or other measures used to manage risks from deliberate exposure or release. They protect bugs from bad people.( iGEM 2019 )
For this part, we updated and revised our risk assessments, we have handed out safety form promptly, to clarify that our lab are Safety Level A laboratories and students are hardly exposed to biological risks, and our project were designed to contribute to the global environment. We carefully analyzed environmental impact for the future application and verified our products will be repeatedly sterilized using heat and germicide during recycled paper production.
We will further explicate our assessments and how and why we implemented biosafety and biosecurity requirements in our project in the following paragraphs.
Our lab follows Chinese legislation about risk 1 biosafety laboratory, and our lab is provided with two autoclaves (respectively for waste sterilization and sterilization for future usage), a ventilator and several laminar flow cabinets with UV sterilization. Besides, our instructors have provided us with a document listing every detail we should pay attention to when we are in the lab.
Our lab has also considered every possibility for us to perform safer experiments, such as using Gel Red, 4S Green Plus nucleic acid dye to reduce toxicity, and equipping Blue Light Gel Imager for our skin and eye protection, etc.
All students including us iGEMers are trying our best to follow all the protocols for better experimental reproducibility.
Besides, our labs strictly follow the rules of garbage classification—household waste and experimental waste are separated, all biowaste containing bacteria are sterilized before disposal. As you can see in the next photos, experimental waste such as tips, syringes, gloves, and plates are collected separately for pollution treatment and waste recycling.
We used the Escherichia coli str. K-12 substr. MG1655, Escherichia coli BL21, and Escherichia coli DH5α, which were provided by Prof. Quan Shu’s Lab and Prof. Wang Qiyao’s Lab, and those three strains are all classified as risk 1 so the harm which can cause to people is minimal. The three types of Escherichia coli (str. K-12 substr. MG1655, BL21, and DH5alpha) we've used, whether as PCR template resource or competent cell, are harmless to human being and surrounding environment. They are both world-widely used engineered bacterium which has been proved to be safe.
Safety by design
Our project aims to construct E. coli (DH5a) which can at first expresses cellulose exoglucanase (cex) and cellulose endoglucanase (cenA) to degrade cellulose into cellobiose, and then the cell expresses cellobiose phosphorylase (cep94A) and cellulose synthase (acsABCD) to further degrade cellobiose into glucose and synthesize bacterial cellulose. Our basic method was co-transforming two plasmids, one pIN2 that constitutively expresses cex and cenA another pIN1 expresses cep94A and acsABCD, as well as adding some regulator genes to achieve automatic switching.
During experiments, we applied some conventional reporter genes like mRFP and GFP for characterizations. All the expressing proteins are non-toxic to both E. coli and human beings.
For fermentation stage and future application, we designed our engineered bacteria to be completely sterilized by heat and UV light. Besides, E. coli is mixed in the product which will be added fungicides and sent to paper mills. E. coli metabolites themselves do not affect the product and bleach is added during the papermaking process. Paper mills will add germicides every step of the way to ensure that the final product will not release any bacteria, which will hugely reduce our impact on the environment.