Our aim is to unite all sectors in order to make a significant societal impact while maintaining responsibility and promoting global welfare. The critical perspectives and feedback from our stakeholders have been instrumental in refining our project plan and solution design into an optimized and feasible system. It is crucial to incorporate the feedback, values, and needs of relevant stakeholders, as individuals often become enamored with their initial designs or inventions, leading to a disregard for valuable criticism and feedback from stakeholders. To prevent becoming overly attached to our initial design, we have implemented a human-centered design approach at every stage through the use of CRISPReporter.
In order to incorporate the requirements of every phase in the design of our biosensor, we have examined the societal, technological, entrepreneurial, and safety aspects of our project through engagement with various stakeholders, including academia, enterprise, government, and the public. Each sector provides a unique perspective on the problem and our solution, resulting in distinct requirements for both our project plan and solution design.
Introduction
Nowadays, biosensors play a critical role in various fields, including biomedical diagnosis, treatment and disease progression monitoring, drug discovery, food control, and environmental monitoring. However, contemporary biosensors cannot record the temporal sequence of analyte events over long durations, which is a crucial requirement for some applications. Therefore, only a long-term sensor that can perform continuous recording would be advantageous for such purposes.
Our solution
After an extensive review of existing literature and brainstorming sessions, we devised a recording system that meets our specifications, which we call a “DNA memory device”. DNA is a high-density storage medium that can be stably preserved and easily accessed using molecular biological techniques. Moreover, gene editing tools can translate signals into DNA modifications, enabling both biosensing and recording. These features inspired us to develop a DNA-based biosensor, CRISPReporter. We envisioned the potential applications and impacts of CRISPReporter in various fields and domains.
CRISPReporter is a novel model for building programmable genetic circuits and developing bioinformatics software in the field of synthetic biology. By integrating CRISPReporter with computational tools, it can serve as an output component for constructing a biological computer.
To evaluate the feasibility and applicability of our design, we consulted with various stakeholders in the relevant fields.
To assess the feasibility and applicability of our design, we conducted consultations with various stakeholders in the relevant fields.
Interactive timeline
Understanding: Determine the line of practice
Our multi-level marker response model was initially conceived to diagnose Inflammatory Bowel Disease (IBD), a condition known for its recurrent nature. However, by communicating with our PI, we learned that the current lack of a validated biosensor model given the complexity of the intestinal environment, led us to dive into a more fundamental field. While this may seem removed from practical applications, it holds immense potential.
After defining the project as a CRISPR-based biosensor, we conducted a public questionnaire survey (See more on the Communication & Education page). We encouraged participants to envision the future possibilities of biosensors. We can see the result below that Medical and health and Environmental have the highest number of votes, coincidentally consistent with our vision. It also makes us more motivated to explore his applications in the diagnostic and environmental fields.
We try to get a deep understanding of the application prospects of CRISPReporter from the public, academia, enterprises, relevant research institutions, government policies, etc.
Reacting and adapting: our solution
Throughout the process of designing and developing our project, we consistently prioritized aligning our biosensor with the needs of stakeholders." Our biosensor was crafted with a continuous awareness of the practical aspects in the field, running in parallel with our laboratory work. We engaged with numerous scientists to gain insight into their methodologies and observed real-world fieldwork with stakeholders. Every time we received feedback on our tool's design, we made it a point to carefully incorporate it into our efforts.
Wet Lab
EL222 directed evolution
To achieve multi-level signal recording, we need to find a system that can accurately simulate signal input. Professor Fei Gan suggested that we use light-sensitive promoters because they are less susceptible to interference than conventional chemical promoters. After consulting Professor Andreas Moglich, we identified three types of light-activated systems: red, blue, and green light promoters.
We discovered two distinct categories of photosensitive components: two-component systems and one-component systems. Through communication with the two professors and literature reviewing, we adopted the blue light-inducible system EL222, a kind of one-component system, as the signal input. It can precisely control the level of gene expression by adjusting the dose or intensity of blue light pulses, while also has a low metabolic burden on bacteria compared with two-component systems.
Furthermore, we decided to evolve EL222 to reduce the promoter leakage and enable it to respond to signals of different intensities, ultimately achieving the goal of accurate recording. Meanwhile, to avoid the continuous self-splicing of the system after responding to the signal, we discussed with Liran Mao and shared the same idea: by controlling the inducer concentration and induction time, we can tune the on and off time of the editing system and achieve intermittent signal recording.
The cascade system
As we aimed to build a comprehensive cascade recording system that precisely captures event sequences, we consulted with numerous experts.
To ascertain the editability of the plasmid and the functionality of our designed system, Xiaowei Liang recommended that we should preliminarily select the CRISPR editing site on genome to verify the feasibility of our knockout system. Professor Ying Zhang highlighted that Lambda homologous recombination technology carries the risk of abnormal recombination during plasmid replication, which could potentially disrupt signal recording. To evaluate the severity of this issue, we designed confirmatory experiments on the system, the details of which can be found on our Results page.
Furthermore, we encountered difficulties in identifying accurate induction conditions to achieve the first-level knockout during our editing induction process. Xingpeng Wen and Liran Mao advised us to refine these conditions and repeat the test, wich led us to construct a matrix of induction times and concentrations to explore optimal induction conditions. Concurrently, Wen suggested that we conduct functional verification of the working feasibility of the CRISPReporter editing system by replacing the Lac constitutive promoter with T7 prior to starting induction. This recommendation has allowed us to refine and enhance the logic of our experimental design.
Dry Lab
In the final iteration of our multi-level editing recording model, the generation of high-quality sgRNA has become an essential component of the project.
Simultaneously, we are confronted with a vast amount of data and uncertainty. The delay between multi-level records and reporting presents new challenges. Given the existing research foundation and the overall characteristics of this field, we have positioned our digital-analog software as a tool that provides “pre-screening and heuristic suggestions for biological experiments”. This approach deeply integrates the specific needs of the project, providing a large quantity of high-quality gRNA for different biological chassis.
Regarding the mathematical model section, Professor Ying Zhang first highlighted the vast diversity of lives which limits reporting accuracy. However, she also acknowledged the efficiency prediction ability of our model. She affirms that we don't need to involve epigenetics in our model, as the genome structure of E. coli is relatively simple. For our project, Zhang suggested prioritizing promoter leakage and homologous recombination during plasmid replication to further improve and optimize the project design. Additionally, Professor Lihua Zhang provided us with valuable insights on method design, reminding us to pay attention to the collection and selection of data sets.
Applications
The CRISPReporter, due to its remarkable recording capacity and potential to sense diverse signals, holds promise for a multitude of applications. We have explored its use in various domains such as diagnosis and environmental monitoring.
Diagnosis
During our visit to the Center for Inflammatory Bowel Disease Research and Diagnosis, we gained insight into the fact that despite continuous updates in the clinical diagnosis and treatment methods of IBD, there remain several shortcomings. Enhancements in IBD diagnosis need to be multi-faceted, encompassing the strengthening of basic research, refining diagnostic criteria, and improving patient education and psychological support.
Dr. Liao, an expert in IBD, expressed significant interest in our project and its potential in IBD pathogenesis recording. He emphasized considerations for transitioning engineered bacteria into practical applications, including the sensitivity of bacterial induction markers and the feasibility of long-term gut colonization. We also discussed targeting specific types of IBD, and reaching an agreement on choosing ulcerative colitis.
Dr. Liao also highlighted the importance of human safety and experimental ethics. Furthermore, Dr. Liao emphasized that our engineered bacteria should not be limited to disease detection but should also incorporate treatment modules to achieve an integration of diagnosis and treatment, thereby facilitating precision medicine. He suggested that our system could be applied to other more common diseases, such as polyp recurrence detection.
To create a substantial impact in this domain, we engaged in collaboration with NWU-China, an iGEM team also dedicated to addressing IBD. We maintained an open channel of communication, exchanging project updates and collectively brainstorming ideas. Our shared commitment to supporting IBD patients and their families led us to distribute an IBD awareness brochure created by NWU-China within our school. We drew inspiration from NWU-China's fundraising initiatives, which encouraged people to provide assistance to IBD patients. The meaningful and inspiring work done by NWU-China resonated with both us and the IBD patient community.
Environment monitoring
Professor Chen expressed considerable interest in our system and highlighted its advantages: it can simultaneously detect several different environmental pollutants and requires less sample pretreatment compared to traditional methods. However, he also pointed out several considerations for transitioning the engineered bacteria into practical application.
- Sensitivity: The accuracy of methods such as mass spectrometry can reach 10-12, thus the sensitivity of this system needs to be determined.
- Cost: Some methods for detecting environmental pollutants are quite mature and cost-effective. Therefore, it is critical that this new method can effectively control costs.
We consulted Professor Chen about the general treatment cycle of pollutants. He explained that the duration varies greatly depending on the medium (water, soil, atmosphere) and the selected methods (physical, chemical, biological). There are also different approaches such as in situ repair and ectopic repair, which add to the complexity. Professor Chen suggested that we could establish a system targeting a new specific pollutant, such as antibiotics, which might facilitate market entry.
Taking Professor Chen’s advice into account, we chose to focus on macrolide antibiotics, a new pollutant. To design a system responsive to macrolides, we referred to the promoter system of 2020 Alto-Helsinki-SINISENS. In the absence of macrolides, downstream genes cannot be expressed. However, once macrolides are present in the sample, the system allows for transcription of downstream genes (see more on our Applications page ).
Bioinformatics
Artificial intelligence has brought significant convenience to biotechnology, and we have experienced this convenience profoundly. This trend has inspired us to envision the future of CRISPReporter, where robots and artificial intelligence could be employed to develop biosensors tailored to our requirements. We envisage that by integrating CRISPReporter with computational tools, it can serve as an output component for constructing a biological computer. However, after consulting with Professor Ying Zhang and Professor Lihua Zhang, we have realized that although our ideas are very meaningful, considering the current factors, we still have a long way to achieve this goal.
Close the feedback loop
Throughout the course of our project, we endeavored to engage with a diverse range of stakeholders within the sector. Our comprehensive perspective of the entire chain led us to an important realization: our tool, originating from a fundamental research project, operates at the base of the chain and possesses the potential to influence all the stakeholders we interact with.
Our interactions with these varied interlocutors offered us invaluable insights into the dynamics of this field and exposed us to a multitude of viewpoints. Despite our pride in our tool, we acknowledge its limitations that need to be considered during its utilization.
In terms of recording accuracy, the probability of recording errors, albeit extremely low, is inevitable. This indicates that there is still scope for optimal improvement in our design. Regarding market applications, we must take into account safety, ethics, cost, and other comprehensive issues to ensure that our projects can be genuinely applied in real life and contribute positively to society. (See more on our Entrepreneurship page)
Our motto encapsulates our ethos: "WHU-China - Work Hard for Unlimited potential of synthetic biology!" The profound human interactions we encountered shed light on the realities of the field and the challenges faced by these stakeholders. We made a concerted effort to incorporate this human aspect into our work.