Overview

We discovered that engineered microorganisms may pose risks in practical application scenarios, leading us to design the Wind-up Cell system. With a focus on biosafety, we conducted a public investigation, researched biosafety regulations, and consulted biosafety experts to ensure the meaningfulness and feasibility of our project. Our project continuously evolves, inspired by expert advice to improve experiments. We have designed a safer whole-cell test paper, which is the current achievable application of the Wind-up Cell system. In the future, we hope the Wind-up Cell system can integrate with other engineering microbial modification methods to enhance biosafety further.

Figure 1. The overview of human-practices.

Why did we create the Wind-up Cell?

With the development of synthetic biology, engineered microorganisms have demonstrated significant potential applications in various fields such as medicine, agriculture, and environmental management. However, in the practical application process, the introduction of engineered microorganisms into these environments may pose potential risks. While engineered microorganisms can serve as drug carriers or directly secrete metabolites in the human body to provide more sustained and gentle treatments, their prolonged presence in the human body may lead to drug resistance or disrupt the existing microbial communities. Additionally, although engineered microorganisms can be utilized for soil remediation, they may also interfere with the microbial diversity in the environment. These potential risks may raise concerns about biosafety, thereby constraining the research and application of engineered microorganisms.

We aim to discover an efficient method to induce the death of engineered microorganisms after they have fulfilled their function, thereby mitigating any potential adverse effects they might pose.

What is the Wind-up Cell?

We have learned that epigenetic modifications can alter the phenotype of specific genes or DNA regions without changing the DNA sequence itself, and the process of removing epigenetic modifications takes some time. Inspired by this, we have designed a system called the "Wind-up Cell."

We combine epigenetic modifications with toxic genes and, in specific induced environments, use epigenetic modifications to suppress the toxic genes, thereby maintaining the cell's survival. Once they leave the specific labortoy environment, the toxic genes are no longer inhibited by epigenetic modifications, initiating a self-destruct process for the microorganisms. During the countdown to the death of the engineered microorganisms, they can perform various functions and die afterward, thus avoiding risks to organisms or the environment.

Is the Wind-up Cell valuable?

We hope to confirm the feasibility and significance of the project from these investigations, and then further clarify the direction of the project and the regulations to be complied with in the research process.

Public biosafety awareness survey

We aim to assess the extent of public awareness regarding biosafety issues. We have designed a biosafety investigation questionnaire, which was disseminated via online platforms and distributed to community residents along with biosafety promotional posters.

In the questionnaire, we provide a detailed explanation of the definition of biosafety and highlight recent noteworthy biosafety incidents both domestically and internationally, along with common biosafety controversies. We present questions to the public to gauge their understanding of biosafety and related laws, and gather their suggestions on how to reduce biosafety incidents and improve biosafety education.

Through this investigation, we have collected public awareness data. The questionnaire results indicate that there is a need for further advancement in biosafety education, and there is widespread recognition of the potential for improving biosafety through advancements in biotechnology. This demonstrates the significance of our project in enhancing biosafety awareness and education. Additionally, these valuable feedback insights will aid us in refining our biosafety promotion and educational efforts, ensuring that our information dissemination is accurate, comprehensive, and meets the needs of the public, ultimately raising public awareness of biosafety.

Biosafety legal research

We consulted the Biosecurity Law of the People's Republic of China and the Biosafety Management Regulations For The Laboratory Of Pathogenic Microorganisms, among other biosafety-related legal documents. This research aimed to understand the regulations we need to comply with in our experiments and resulted in a research report. We found that our experiments fully adhere to the relevant legal requirements and are permissible.

Consultation with biosafety expert

Weiwen Zhang
Center for Biosafety Research and Strategy, Tianjin

We consulted Professor Zhang Weiwen, an expert in the field of biosafety, regarding our project and questions related to biosafety regulations. Professor Zhang has been involved in the formulation of the Tianjin Biosecurity Guidelines for Codes of Conduct for Scientists and is also a member of the iGEM Biosafety Committee.

Professor Weiwen Zhang acknowledged the biosafety significance of our project. Regarding the application scenarios of our project, Professor Zhang suggested that we should focus on more prominent issues. This way, our Wind-up Cell system can maximize its value and become more easily understood and accepted by the general public.

Furthermore, in terms of biosafety regulations, Professor Zhang pointed out that the current biosafety review process is not comprehensive enough and has certain shortcomings in practical supervision.

After considering Professor Weiwen Zhang's suggestions and engaging in discussions, we have reevaluated the potential application scenarios of the project. In addition to envisioning future applications in fields such as gut microbiota therapy and marine oil degradation, we are also exploring immediate applications feasible with the Wind-up Cell system. Simultaneously, in the context of existing biosafety regulations,we aspire to utilize the Wind-up Cell system to enhance biosafety standards and advocate for the establishment of unified guidelines for the application of engineered microorganisms. This will facilitate the enhancement of regulatory processes in the future.

Experimental feedback

After clarifying the project's value and direction, we plan to construct the Wind-up Cell system in both prokaryotic and eukaryotic organisms. With the assistance of professors such as Yi Wu and Yingxiu Cao, our project is progressing gradually.

Eukaryotic organisms

: After reviewing the literature, we constructed a fusion protein of dCas9 and histone deacetylase in yeast to achieve the suppression of toxic genes.
Challenge:
The suppression tool was not effectively inhibiting the expression of toxic genes.
Solution:
We consulted with graduate student Yuqi Wang and doctoral student Yali Cui, who specialize in yeast research, in hopes of gaining some advice.

Yuqi Wang: Postgraduate, Tianjin University
Advice
Wang:
The achievement of silencing effects is often the result of the combined action of multiple factors. To enhance the silencing effect on toxic genes, one might consider employing multiple gRNAs to target simultaneously.

Implementation
We designed multiple sgRNAs targeting the toxic gene upstream of promoter and open reading frames.

Yali Cui: Doctoral Student, Tianjin University
Advice
Cui:
HML can recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci.

Implementation
Inspired by her, we conducted further research and decided to add HML sequences to both ends of the toxic gene. These two sequences can recruit silencing factors, aiming to achieve a stronger inhibitory effect.

Result: The inhibitory effect of dCas9 fused with multiple sgRNAs and histone deacetylase fusion protein has been further enhanced. The addition of HML sequences to both ends of the toxic gene resulted in a significant improvement in suppression. However, we currently lack a method to release the inhibition caused by HML sequences. Further exploration will be conducted to improve the potential of this suppression tool.

Prokaryotic organisms

: After reviewing the literature, we employed Dam methyltransferase to methylate the promoter of the toxic gene (mioc) in E. coli to achieve the suppression of the toxic gene.
Challenge:
The suppression tool proved ineffective in repressing the expression of the toxic gene.
Solution:
We decided to consult Associate Professor Yingxiu Cao, a long-time expert in E. coli research, in hopes of obtaining some insights and recommendations.

Yingxiu Cao: Associate Professor and Master's supervisor, School of Chemical Engineering, Tianjin University
Advice
Cao:
If using Dam methyltransferase alone yields limited results, you can combine it with the CRISPRi system to enhance the specificity of Dam methyltransferase, thereby strengthening the suppression of the toxic gene. You can further consult Professor Yi Wu on how to design a rational and effective CRISPRi system and integrate it with Dam methyltransferase.

Implementation
After receiving Professor Yingxiu Cao's advice, following an internal team discussion, we have designed two approaches:
1. Dam methyltransferase and the CRISPRi system are separately constructed in two plasmids and then transferred into the same bacterium.
2. Construct a fusion protein of dCas9 and Dam.
Regarding the feasibility of these two approaches, we are consulting Professor Yi Wu for guidance.

Yi Wu: Professor and Doctoral Supervisor, School of Chemical Engineering, Tianin University
Advice
Wu:
Achieving co-transformation with dual plasmids presents greater difficulties, making the approach of constructing a fusion protein of dCas9 and Dam more feasible.

Implementation
After our initial attempts with both systems and considering Professor Yi Wu's advice, we have decided to proceed with approach two, which involves constructing a fusion protein of dCas9 and Dam. With the guidance of sgRNA, this fusion protein will jointly target the desired sequence to achieve the inhibitory effect at the target site.

Result: The effectiveness of the suppression tool has significantly improved.

The applications of the Wind-up Cell

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. This toolkit can be adapted to various application scenarios of engineered microorganisms, is expected to reduce the application risk of engineered microorganisms and clear the way for new synthetic biology technologies to be put into practice, thus bringing revolutionary changes to the development of synthetic biology technology.

In terms of currently achievable applications, due to the controllable activation capability of the Wind-up Cell, which initiates a countdown to cell death, we have designed a safer whole-cell test paper for substance detection.

For further details on the future application of our project, please refer to the Implementation Page.

Introduction to the test paper

The fundamental structure of the test paper includes the engineered microorganisms with Wind-up Cell system and a solution containing inducers. When needed, the user retrieves the test paper from the solution, triggering the self-destruct mechanism of the Wind-up Cell. This deactivates the gene suppression tools, leading to the expression of toxic genes, and initiates a countdown to cell death. In this state, we can analyze the substance under examination by observing the intensity of the fluorescent protein on the test paper. Upon completing the test, the engineered microorganisms with Wind-up Cell system on the test paper inevitably undergo cell death. You can gain a clearer understanding of the basic principles of the test paper through our video.

Envisioned applications of the test paper

We anticipate that this type of test paper can be further improved and applied in people's daily lives in the future. It has the potential to meet the needs of both the general public and professionals. For example, in outdoor settings, it can assist individuals in quickly detecting the presence of heavy metals in water or determining if household food has spoiled, among other applications.

Investigation on the application of the test paper

To gauge public opinion and acceptance of this whole-cell test paper, we have designed a survey questionnaire. The questionnaire provides an introduction to the potential risks associated with engineered microorganisms escape and demonstrates the application principles of the test paper. We inquire about respondents' opinions regarding the test paper and their willingness to use it. The survey results offer us valuable public feedback and suggestions, as well as new insights into expanding the applications of the test paper.

In the future, we will further improve our kill switch and associate the fluorescence detection signal with the mobile APP, which will not only improve the detection intensity of the fluorescence signal, but also facilitate the reading and analysis of the detection results, and produce a more convenient and efficient whole-cell test paper detection system.