Overview

With the development of synthetic biology, engineered live microorganisms shows broad development prospects in many fields because of its unique advantages. However, this inevitably requires living engineered microorganisms to leave the conventional laboratory culture environment and enter the actual application environment such as the human body, factory, soil, water body, etc. It is difficult to carry out comprehensive sterilization after playing a role, thus causing the risk of engineered microorganisms spreading in the environment, and may cause potential impacts on the environment and human health. In the next 10 years, the development of biotechnology will be more rapid, and it will be more closely integrated with production and life^[1], which makes people urgently need a more effective technology to prevent the potential risks brought by the spread of engineered microorganisms in the environment. To this end, iGEM Tianjin combined Human Practices research results to carry out design iterations, developed Wind-up Cell, a novel biocontainment composed of epigenetic modification tools and toxic genes, and combined this kill switch with substance detection to create a safer whole-cell biosensor as a test paper. Our new technology not only protects humans and nature but also protects the more stable development and application of synthetic biology technology.

Figure 1. Concept of our project

Background

Application status of living engineered microorganism

In terms of medical care and health,on the one hand, engineered microorganisms can be used as a carrier to transport drugs to diseased tissues or organs, avoiding the first-pass effect of drugs, thus greatly improving the therapeutic effect^[2]. On the other hand, engineered microorganisms can express enzymes that degrade related metabolites, so that we can achieve more sustainable and convenient treatment for metabolic diseases such as phenylketonuria and diabetes ^[3][4]. In addition, studies have found that bacteria can selectively penetrate tumor sites by utilizing the tropism of bacteria for tumor microenvironmental features, which may solve the problem of limited penetration of traditional injected drugs into tumors^[5], and may give people new hope for overcoming cancer.

In terms of industrial production, living engineered microorganisms can be used to produce high-value-added products such as antibiotics, paclitaxel, and ginsenosides. It may solve the problem of insufficient natural production of these compounds and meet people's needs.

In terms of the environment, as early as the end of the last century, researchers began to try to construct engineered microorganisms to deal with oil pollution in the ocean. This method is low cost and does not cause secondary pollution^[6]. Later, researchers have verified the feasibility of microbial remediation of pesticide-contaminated soil in large-scale experiments, and have constantly tried to build multi-functional and efficient degraded engineered microorganisms^[7]. In addition, engineered microorganisms may also be able to effectively repair water and soil polluted by heavy metals^[8]. Today, the world has formed several professional companies, such as BCI and Engineering Services and Bio-Remediation Company in America or ECET in Japan. Moreover, a series of engineering bactericide products such as OleoBact have been launched for environmental remediation^[9].

Figure 2. Application status of living engineered microorganism

Application risks of living engineered microorganisms

Through the previous Human Practices research, we found that although engineered microorganisms have broad application prospects in the above fields, many valuable application directions have not been approved for large-scale practical application due to many worrying potential risks in practical application scenarios. In terms of medical treatment, we need engineered microorganisms to continuously and stably express related products, but there are natural microbiomes in human organs such as tumors and intestines, and the long-term existence of engineered microorganisms may induce local microbiome disturbances. In addition, the long-term existence of engineered microorganisms's drug-resistance genes in humans may also produce drug resistance in vivo^[10]. In the field of industry and environment, the long-term survival of engineered microorganisms in the environment may interfere with the original microbial diversity and natural ecological balance of the environment. According to relevant literature reports, when Ford added Pseudomonas fluorescens containing the lux reporter gene to soil contaminated with naphthalene, phenanthrene, and anthracene for in-situ repair, a small number of engineered microorganisms escaped out of the test environment^[11]. Top et al. found that E.coli with the czc gene which is resistant to cobalt, cadmium, and zinc can be conjugated to Alcaligenes eutrophica under experimental conditions. Moreover the frequency of conjugation is 1.5×10^-8 to 1.5×10^-6[12].

At the same time, these potential risks will cause concern and limit the application potential of engineered live microorganisms, which will affect the development of engineered microorganic therapies, the production of new natural compounds, and the effective treatment of environmental pollution. It has greatly limited the application and development of synthetic biology technology and the progress of human civilization.

Figure 3. Application risks of living engineered microorganism

What we have done

To address these issues, iGEM Tianjin designed a novel kill switch consisting of Eepigenetic modification tools and toxic genes and iterated the design continuously through Human Practices. The microbes equipped with this kill switch we designed are called "Wind-up cells." The epigenesis tool of Wind-up cells inhibit the expression of toxic genes in the culture condition with specific inducers. When it leaves this particular culture environment, its winding key is released, the kill switch is activated, relieving the inhibition on toxic gene, which are then transcribed and translated to generate toxic proteins. After a certain duration, the cell undergoes cell death.

Figure 4. Novel kill switch

Figure 5. The working principle of Wind-up cell

Furthermore, iGEM Tianjin combines this kill switch with substance detection, creating a safer whole-cell biosensor as test paper. When we need to use this test paper, the wind-up cell's kill switch would be activated immediately, initiating a countdown to cell death. Meanwhile, we could use them for detection of specific substance. The detected substance induces downstream gene expression, enabling the cells to express an abundant amount of fluorescent proteins. After completing the detection function, the wind-up cells on the test paper proceed toward an inevitable death.

Figure 6. The working principle of our test paper

Advantages

Our wind-up cell is safe. On one hand, the toxic genes constitutively expressed are only suppressed in specific environments. Upon leaving the designated environment, without the need for additional inducers, the toxic genes are expressed, causing cell death. On the other hand, our project is based on epigenetic modifications that suppress the expression of toxic genes. These epigenetic modifications are heritable, and as cells undergo generations, the removal of epigenetic modifications induces a delayed death effect in cells. During this period of delayed death, cells exert specific functions, providing a novel application scenario for synthetic biology. This ensures that engineered microorganisms cannot persist in the natural environment, thus effectively preventing the environmental risks associated with genetically engineered microorganisms.

Future

Through HP's interview with Professor Weiwen Zhang, an expert in the field of biosecurity, we have more prospects for the project. In the future, on the one hand, we will further improve our kill switch, and associate the fluorescence detection signal with the mobile phone APP to make a more convenient and efficient whole cell test paper detection system. On the other hand, our project will replace different toxic genes and combine them with various epigenetic modification tools to develop a new series of kill switches and organize a kill switch toolkit that can freely regulate the survival time of engineered microorganisms. Thus, our project can significantly reduce the risk of the spread of genetically engineered microorganisms in the environment, reducing their potential threats to the ecological environment and human health. At the same time, our project can also make synthetic biology technology can better accept the United Nations "Cartagena Biosafety Protocol", as well as the biosafety management regulations of the EU, China and other countries, while reducing biosafety risks, reduce people's concerns about synthetic biology technology, and contribute to the safer and more stable development of synthetic biology technology.

Figure 7. Associate with the mobile phone APP

Figure 8. Kill switch toolkit