Team:CAU China/Safety
Safety
In our project, safety is indeed a big deal, no matter in project design or daily lab work. However, we’ve also learned that there is no absolute safety in any bio-projects and the vital thing iGEM expects us to do is to assess and manage the possible risks, and to review and revise our safety measures as project evolves.
To achieve this, we design an overall “Safety Framework” to identify possible risks logically and strive to minimize them in the following work.
“Safety Framework”
This set of procedures can roughly be divided into two parts: Safe Project Design & Safe Lab Work.
In Safe Project Design, the first thing we should consider is the safety of our aimed products. If what we need is harmful to human body or environment, we need to rethink about the aim of our project. Following that, we should choose a chassis that lives up to our engineering needs and is non-pathogenic. In environmental projects particularly, this chassis should not harm the nature as well. In addition, to achieve our engineering goal, we should consider about the safety of the parts we will use and pay special attention to toxins involved. Finally, we should consider whether induced lethality is necessary in our project and design a suitable kill switch to avoid escape or competition with natural bacteria.
Plus, we reckon human practice as a crucial part in safety and security, too. As undergraduates, we are likely to underestimate dangers due to lack of experience. So we think our risk management would improve if we get security advice from professors, understand local restrictions about the use of bio-products through entrepreneurship, and learn enough “safe use” examples by communication.
After carefully considering the project design, we will be devoted to wet lab work. When it comes to Safe Lab Work, we should firstly develop a set of lab rules that everyone in this team should obey and discuss the necessary supervision and punishment policies. However, we can not promise all going well and we need to pre-plan about accidents, including clarifying what protection we have in lab and arranging regular safety check to recognize dangers as soon as possible.
There are two check points in this set of procedures to help us review and revise. The first lies in front of the lab work to remind us to recheck the safety of design before doing any experiments, and the second is the regular safety check.
To sum up, this framework begins with the ultimate aim of the project, builds up according to the design logic, suggests bringing in human practices in safety consideration, and involves essential elements in safe lab work.
We will present how we use this framework in our project below and we hope our “Safety Framework” can inspire future iGEM teams to better manage safety and security issues.
Safe Project Design
Products We Need
Our aimed product is γ-polyglutamic acid (γ-PGA), which is a viscous polymer formed by D and L-glutamic acid. Research has shown that γ-PGA does no harm to human body and the environment (Park Sung-Bin et al. 2021). As a matter of fact, γ-PGA is initially found in natto, a kind of food, and is widely used as an absorbent in cosmetics industry, which is a ready proof for its safety.
According to literature, some bacteria, including Bacillus subtilis in soil, can naturally degrade excess γ-PGA at a slow rate (Chen et al. 2008). The degradation product is glutamic acid, which is a common amino acid that can also promote soil microenvironment. So even though our engineered bacteria produced excessive γ-PGA，it would generally be degraded and do no harm to soil at all.
Bacteria We Choose
The chassis we choose is Corynebacterium glutamicum ATCC 13032, which is non-pathogenic, doesn’t produce endotoxin itself, and has been widely used in previous iGEM works (e.g. JNU-China, 2019) and fermentation researches.
Apart from that, we also used Bacillus subtilis strain 168 to get PGA-producing genes capA/capB/capC and Escherichia coli DH5α to amplify plasmids. Both of the bacteria are commonly used in labs and do little harm to human body with adequate protection.
Parts We Use
Our project contains genes coding for polyglutamate synthases and enzymes in TCA cycle (ODHC, PEPC). These protein products are totally from natural physiological activity and do no harm to human, plants, bacteria and soil environment.
Besides, our project also involves endoribonuclease toxin ndoA (also called mazF). It can kill the bacteria by cleavage of the mRNA and thus is not toxic to the environment and other animals. Additionally, as our experiments Results have shown, the CFU has a slow increase after the first few killing hours. Hence, we assume that when ndoA kills the bacteria and is then released into the soil, it will soon be broken down to amino acids and won’t cause any harm to other bacteria.
The plasmids we use, pXMJ19 and pK18mobsacB, are harmless to human as well. Since we plan to insert the genes into the genome, no antibiotics will be put into the soil and will do no harm to non-resistant soil bacteria.
Kill Switch We Design
Proper kill switch design is a necessity to our project, because the proposed implementation of SSR is a kind of microbial agent that will be put into the saline-alkaline soil to restore it, bringing in problems about excessive multiplication and competition.
We design a kill switch that will be turned on particularly when both the ionic strength and pH of the soil decrease to an expected level. In other words, when our engineered bacteria complete the mission of restoring the saline-alkaline land, it will die automatically due to the failure of repression of the toxin ndoA.
Considering that the normal environment around us doesn’t have high ionic strength and pH neither, we believe that this kill switch design applies to possible escape of lab bacteria as well.
You can know more about our approaches in Design.
Advice We Gain
We consulted the manager of Sino Green Agri-Biotech, Lixia Zhang, for bio-safety limitations about microbial agent in China and possible solutions.
Ms. Zhang told us that any use of engineered bacteria in natural environment is still strictly prohibited in China, but the government will not ban legal scientific studies of them. She emphasized the necessity of “Do Not Release Policy” and suggested stricter control in induced lethality. She also shared some microbial agent products in their company to illustrate how they follow the bio-safety rules and how much efforts they devote into security management.
In addition, we asked the public for opinions on the application of synthetic biology products like golden rice. Most of those surveyed approved of its nutrition value, while they also clarified that they wouldn’t give it a mere try until it has been proved to be 100% non-toxic. Also, some expressed concern about the transgenic products and said it is only the last choice. Particularly, one participant said that he/she strongly opposed the golden rice because he/she heard that the research group take Chinese children in poor areas as guinea pig, indicating the nationality and political concerns.
When it comes to environmental products, the public shows more tolerance. They said they would allow the release of synthetic bio-products as long as they passed certain assessments and showed no harm to the food chain.
These inspirations we gain from human practices are useful supplements for our safety & security work. You can see more information in Human_Practices
Safe Lab Work
Rules We Obey
Before starting, we received general laboratory safety training from our PI and instructors, including how to handle emergencies in lab work, the common first aid treatments, the waste disposition and the position and limitations of dangerous reagents in this lab.
Next, with the suggestions from instructors, we drafted detailed lab safety rules particularly for our project:
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Based on that, we picked out 5 rules that are most related to daily lab work and wrote them on a board in lab to remind us from time to time.
We also developed the supervision and punishment policies to reinforce the awareness of safety. For 7 days in a week, there will be a student leader on duty of supervision. He/She should check the tidiness and safety of the lab at the end of that day and finish the necessary cleaning. Besides, he/she should also report and record any dangers or abnormal operations done by teammates. If a team member has done wrong too many times, he/she will be banned from lab work for 3 days to re-learn about the safety rules and be responsible for cleaning for the rest of the week.
Protection We Have
The major lab we are working in is a biological safety cabinet (BSC) which is equipped with UV sterilizers and high efficiency particulate air filters to protect lab workers and the environment from biohazards.
Besides, our lab provides us with necessary protections, including evacuation exit, emergency eye-wash and shower units, fire extinguisher, smoke alarm, masks, gloves, lab overall and skin disinfectant. For COVID-19 particularly, there are daily check of body temperature, spare N95 masks and routine disinfection of the environment.
Regular Safety Check
The lab we are working in has a senior experimenter, Prof. Wang Baoqing, as security chief. He will check the security in our lab carefully with other administrators every two weeks, in case we are careless about safety and overlook underlying dangers.
Reference:
[1] Park Sung-Bin et al. Poly(glutamic acid): Production, composites, and medical applications of the next-generation biopolymer[J]. Progress in Polymer Science, 2021, 113
[2] Chen et al. Degradability of poly-γ-glutamic acid in environment[J]. Environmental Science and Technology, 2008, 31(11)