Basic Knowledge

1. Chain Substitution Reaction

Chain replacement reaction is a process in which a nucleic acid double chain is combined with an exogenous molecule and replaces one of the original chains. In this process, the original chain is squeezed out by the exogenous target molecule to form a new and more stable complex structure.

2. CRISPR-Cas12a System

CRISPR-Cas12a, a member of the Class II V CRISPR-Cas system, is a multi-domain protein composed of approximately 1,300 amino acids. Guided by crRNA, it recognizes target sequences and binds to Cas12a. The Cas12a-crRNA complex then identifies and attaches to the target DNA, causing significant conformational changes that activate a nonspecific nuclease site. This triggers trans-cleavage activity, enabling rapid indiscriminate degradation of any single-stranded DNA in the surrounding environment.

3. Use of Chain Replacement Reaction and CRISPR-Cas12a in Our Project

This project focuses on developing a highly sensitive biosensor for detecting antibiotic residues. The sensor consists of an identification module and a signal amplification module. The signal recognition module employs aptamer design, where aptamer sequences bind to antibiotic molecules in the solution system, causing changes in the double-stranded structure and releasing a short strand. Two feasible approaches are designed for the signal amplification module: one utilizes the released short strand sequence to bind with downstream T7 promoters, initiating transcription and amplifying signals through fluorescently labeled transcripts; the other leverages Cas12a's trans-cleavage activity using short ssDNA "reporters" tagged with both fluorescent and quencher groups at both ends. When intact, the reporter quenches fluorescence; upon activation of Cas12a, trans-cleavage occurs, immediately generating detectable fluorescent signals.

4. T7 Promoter

The T7 promoter, a 17 nt conserved sequence (5′-TAATACGACTCACTATA-3′), is highly specific and efficient for recognition by T7 RNA polymerase, enabling immediate initiation of downstream DNA transcription. Unlike cellular RNA polymerases, T7 enzymes are not subject to host regulation, allowing rapid transcription rates — each template can produce hundreds to thousands of RNA molecules within minutes. This project will leverage these high-efficiency transcriptional properties to design a signal amplification strategy.

5. Conclusions

The aptamer-based biosensor achieves specific recognition of antibiotic molecules. The sensor's sensitivity is enhanced by a signal amplification module based on T7 promoter rapid transcription and Cas12a trans-cleavage activity.

Education

As passionate and responsible explorers of science, our goal goes beyond developing a technology. We see our project as an opportunity to engage in dialogue with the world, to listen to the voices of the public, and to ensure that our work brings meaningful benefits to society and to humanity.

Human practices play a vital role in shaping and advancing our project. Inspired by our investigation into antibiotic usage in animal husbandry, we engaged in conversations with experts and industries to reflect on practical needs and potential shortcomings. This process guided us in overcoming technical challenges and identifying directions for improvement.

Our long-term vision at HunanU includes a rural education program dedicated to igniting the spark of science in resource-limited areas. We encourage children to improve their lives through knowledge and science. In these activities, we focus on food safety, teaching children about the risks of antibiotic residues through themed lectures, role-playing activities, and quiz games to promote healthier lifestyles. Beyond classrooms, we also work with local communities—holding workshops with farmers to better understand agricultural antibiotic use while promoting safe and responsible practices, establishing genuine two-way communication.

Beyond our own project, we strive to bring synthetic biology closer to the wider public. In science outreach events held in Changsha, participants were invited to build gene circuit models themselves. During our synthetic biology summer camp, we conveyed the core idea of the field—“modular design and functional creation”—through engaging and interactive activities, inspiring children to imagine the fascinating world of synthetic biology.

We believe that science communication in synthetic biology is not only about sharing knowledge but also about cultivating the next generation of enthusiasts and contributors. In 2024, we established the HunanU iGEM Club, where some members have now become core members of the 2025 team. This year, through lectures, lab experiences, and more, we continue to pass on our passion for synthetic biology and iGEM, keeping the flame alive across generations.

Safety

Our team implemented safety principles at the technical source: By adopting a cell-free, life-free, and vector-free in vitro system, we used only static oligonucleotides with dozens of bases to eliminate potential pathogen transmission, gene escape, and ecological accumulation in a single step. Simultaneously, we systematically identified and addressed chemical risks such as antibiotics, DNA, and enzymes, pre-setting countermeasures for each operational phase. This approach achieved intrinsic safety from "design" to "application".

At the laboratory facility, physical barriers were established through Personal Protective Equipment (PPE), fire suppression and emergency sprinklers, along with annual sterilization equipment. The protocol followed a "train before work" approach: operators first received training before entering the lab, then performed cabinet operations, and neutralized liquid waste with chlorine-based disinfectants. All waste materials must be properly classified and decontaminated before leaving the lab. This closed-loop system not only safeguarded personnel but also effectively isolated environmental stressors like antibiotics before discharge, ensuring comprehensive containment of potential hazards.

Parts

We designed multiple aptamer-based sensors for detecting vancomycin and tested them one by one, selected the sequence of the best-performing sensor, and added it to the registry.