Risk Identification

Agriculture-related research involves application in environment and human exposure, so it needs to bear the corresponding biosafety risks. Based on what we know, biosafety risks refer to the potential threats posed by biological substances to the health of living organisms, primarily humans, and the environment. Therefore, we need to take safety measures with respect to the effects of biological research on humans and the environment

Here, we will provide safety principles from three aspects, including experimental, technical and practical. Thus, we prevent the risk of exposing people to toxins and modified sequences. In addition, we tried to reduce horizontal gene transfer after our product is applied to the environment, which can reduce ecological risk.

Before we began our project, we first analyzed the potential risks we might encounter. And we finally came to the conclusion that our risks mainly came from three parts: 1) the organism we use; 2) the lab activities; 3) risks during implementation.

This year, we choose a quite normal organism in the environment, Agrobacterium tumefaciens, as our chassis. According to the check-in form, Agrobacterium tumefaciens can be classified as Risk Level 1 organisms, which means that it has low risks of affecting our health. Therefore, we assess that the overall risks are low. However, in order to reduce horizontal gene transfer and avoid leaking out, we still find it necessary to design a kill switch for our engineered bacteria.

Of course, our lab activities can exert a certain degree of security risk, which we must try to avoid.

We use Agrobacterium and Tobacco mosaic virus as transformation vectors. These vectors may pose a risk to plants in the environment.

We will be growing tobacco within the laboratory, and their seeds and pollen may cause allergies among laboratory members.

Management

Safety Design

Thanks to the self-amplifying system carried on our RNA, our RNA can reach a threshold that can produce an effect by self-amplification instead of relying on the growth and reproduction of agrobacteria. So, agribacterium's mission is done after the transient transfection is completed, which means we do not require agrobacterium to survive in the environment for a long time. Considering our engineered bacteria are mainly used in plants, and plants need sunlight to grow, so we chose sunlight as the way to initiate agrobacterium's suicide.

As for the gene circuit, we introduced a constitutive promoter expressed in Agrobacterium and a phototoxic protein, trying to ensure the biosafety of our project.

Different from our self-amplifying RNA, this sunlight-controlled suicide switch is not on the T-DNA of Agrobacterium, so it will not be transfected into plants by Agrobacterium, which makes the two parts not influence each other and reserves Agrobacterium's ability to commit suicide.

Light induced kill switch

We first chose KillerRed that had been registered in the iGEM Parts ( BBa_K1184000 ) to commit suicide. According to research, KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) under yellow-green light (540-585 nm) and can be used as a kill switch for biosafety applications.

In addition, We synthesized the optimized mini singlet oxygen generator protein (miniSOG) gene, This is a phototoxic protein derived from the LOV protein[1]. Upon illumination, miniSOG generates sufficient singlet oxygen substances to kill bacteria[2]. In order to use this protein in Agrobacterium, we optimized its codon.

Promoter

We chose the constitutive promoter 50Spro. This promoter controls the expression of the gene rpmB in Agrobacterium tumefaciens str. C58, which encodes the 50S ribosomal protein L28[3].

Figure 1: The gene circuit design and protein functions of safety module

Through light experiments, we showed that although sunlight conditions can affect the suicidal effect of engineered bacteria, the viability of the strains carrying phototoxic proteins was significantly reduced after sunlight exposure on the plate.

Figure 2: Number of colonies after light treatment at different times on a sunny day

Lab Regulation & Safety Trainning

In order to reinforce the management of the labs, the official of our university delivered management regulations. All of our activities are instructed under the official guidance. Besides, our lab also special regulations on management of instruments and equipment, which we must obey during experiments.

To gain access to our lab, we must apply on the lab website and take a test. The test included basic experiment skills, lab cleaning, experimental waste disposal, ethics, chemical reagents storage, lab rules, equipment safety, and emergency measures. Only those who pass the test are allowed to carry out experiments in our lab.

Associate Professor Yang Fan is the instructor of our lab. She has been in charge of our lab for 8 years. She is familiar with most molecule cloning experiments and can offer suggestions at any time. So our experiment specification is under the control of the instructor in order to avoid any possible danger. Before we began our business in the lab, we also had a 'safety education class' delivered by her to train us the specific rules to follow in the lab and how to deal with the possible emergency. When we are carrying out our experiments, our instructor will supervise our experimental operations,decreasing the posibility of unnecessary danger and damages.

Figure 3: A glimpse of our experiment bench

Figure 4: The clean bench for sterile operation

Our laboratory has a dedicated lab instructor, who trains us on laboratory procedures, reviews our experimental plans, and ensures compliance with laboratory safety requirements. In addition, ourteam members are vigilant about the general biosafety standards of synthetic biology, consult literature to design more efficient safety modules, and regularly communicate with our mentor todiscuss and optimize product safety.

Our work is carried out in strict accordance with the relevant laws and regulations and campus safety rules.

Ministry of Education of the People's Republic of China has released the "Safety Regulations for Higher Education Laboratory" and distributed it to all higher education institutions.[4]

At Zhejiang University, the Laboratory Equipment and Management Department is responsible for the supervision and safety management of all laboratories.[5]

Implementation Risk Management

Safety for Animals, Plants, and the Environment

Because our biosafety module and the working module are located in the same plasmid, it reduces the viability of engineered bacteria in the wild and limits horizontal gene transfer, thereby mitigating impact on environmental microbial communities.[6] Moreover, compared to traditional chemical pesticides, The biodegradable nature of RNA reduces the risk of exposure to the environment.

Our challenge

(1) Suicide of Agrobacterium may be affected by environmental factors and the effect is unstable. Agrobacterium has different physiological activities under different temperature and humidity conditions, so it cannot be used in all agricultural areas.

(2) Trade-off Between Yield and Resistance: There is often a trade-off between yield and disease resistance. Energy and resources that a plant could use to defend against diseases are instead used for growth and reproduction. Therefore, the use period and amount of engineered bacteria need to be strictly controlled.

Before the engineered bacteria are officially released into the environment, rigorous biosafety test in accordance with the requirements of national governments and relevant departments will be conducted. This is to ensure that the engineered bacteria and related biological components will not cause harm to the environment. Our team will also work under the guidance of technical personnel to modify the biolocical components and optimize them to be harmless to the environment, thus minimizing any potential risks. Complete and reliable experimental data will be included in the preparation of the application documents for approval, thus providing the basis for patent application and application approval.

Reference

[1] Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 2011 Apr;9(4):e1001041.

[2] Rubén Ruiz-González, John H. White, Aitziber L. Cortajarena, Montserrat Agut, Santi Nonell, Cristina Flors, "Fluorescent proteins as singlet oxygen photosensitizers: mechanistic studies in photodynamic inactivation of bacteria," Proc. SPIE 8596, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications V, 859609 (21 February 2013).

[3] 徐丽萍. 基于荧光报告载体的农杆菌atu4860基因启动子调控机理研究[D].扬州大学,2017.

[4] http://www.moe.gov.cn/srcsite/A16/moe_784/202302/t20230220_1045998.html

[5] http://zjulab.zju.edu.cn/39425/list.htm

[6] Guo M, Ye J, Gao D, Xu N, Yang J. Agrobacterium-mediated horizontal gene transfer: Mechanism, biotechnological application, potential risk and forestalling strategy. Biotechnol Adv. 2019 Jan-Feb;37(1):259-270. doi: 10.1016/j.biotechadv.2018.12.008. Epub 2018 Dec 21. PMID: 30579929.