Safety and Security Overview

Compliance with iGEM Rules and Policies

Our project strictly adheres to iGEM’s safety and security policies, ensuring all research activities are conducted responsibly and with utmost care for safety.

  1. Prohibited Activities
    • We do not engage in any prohibited activities, such as using organisms from Risk Group 3 or 4, releasing GMOs outside the lab, or conducting human testing.
  2. iGEM White List Compliance
    • Our project complies with the iGEM White List, ensuring all organisms and components used are approved, without requiring special permissions.
  3. Advance Permission Requirements
    • We avoid any activities requiring advance permissions, such as working with animals or conducting experiments involving human samples.
  4. Human Subjects Research
    • We are not conducting human subjects research that would require formal ethics approval.

Lab Details

  1. Lab Work and Access
    • Our project is conducted in Biosafety Level 1 and Level 2 labs, ensuring proper containment measures are in place for all experimental procedures.
    • Lab Photos: [Insert photos of lab space and safety features.]
  2. Biosafety Level
    • We use both Level 1 and Level 2 labs to ensure safety in handling different types of biological materials.
  3. Work Areas
    • Open Bench: For routine lab work.
    • Biosafety Cabinet: For tasks that require containment.
    • Clean Bench: Used for molecular cloning to maintain a sterile environment.

Safe Project Design

  1. Chassis and Parts Selection
    • We selected non-pathogenic E. coli strains DH5α and BL21 due to their suitability for lab use, ensuring the safety of our experiments.
  2. In Vitro Testing
    • All experiments are conducted in vitro, maintaining a controlled and safe environment.
  3. Molecular Cloning and Protein Expression
    • Our project involves transforming E. coli DH5α with plasmids containing genes for split protease and its substrate. E. coli BL21 is used to express target proteins, such as PPVp fused with FRB and FKBP binding rapamycin, and truncated GFP-linker (PPVpsite)-truncated YFP.
  4. Safety Mechanisms
    • We incorporate biosafety features such as “kill-switches” and use non-toxic, well-characterized parts to enhance safety in our design.

Safe Lab Work

  1. Daily Lab Procedures
    • Our team follows strict safety protocols, including the use of PPE, proper waste disposal, and equipment sterilization.
  2. Risk Assessments
    • We conduct comprehensive risk assessments to identify potential hazards and implement mitigation strategies, especially in molecular cloning and protein expression experiments.

Ethical Considerations

  1. Responsible Research
    • Our project is conducted ethically, aligned with local and global safety standards.
  2. Engagement with Safety Policies
    • We actively follow iGEM’s safety guidelines, ensuring compliance across all aspects of our project.

Anticipating Future Risks

  1. Real-World Application
    • Our project focuses on developing a cellular communication system, primarily for lab use. However, potential industrial or therapeutic applications may be explored in the future.
  2. Release Beyond Containment
    • No release beyond containment is anticipated. If future development requires field trials or therapeutic applications, we will seek regulatory approval and conduct appropriate reviews.
  3. Potential Adverse Outcomes
    • We have considered potential risks, including harm to human health, agriculture, or the environment. While the current scope minimizes these risks, future developments will be carefully managed to prevent negative outcomes.
  4. Preventing Autonomous Spread
    • Our engineered organisms are not designed for autonomous spread. We employ biocontainment strategies, such as “kill-switches” and auxotrophy, to prevent unintended release.

Managing Risks

  1. Expert Support
    • We receive guidance from institutional biosafety officers and experts in risk management to address any potential hazards.
  2. Safety and Security Rules
    • Our work complies with national and institutional biosafety regulations, ensuring adherence to [Insert relevant regulations].
  3. Additional Support and Review
    • While no additional support is currently required, future developments will involve regulatory reviews and expert consultations.
  4. Safety and Security Training
    • All team members have undergone thorough safety and security training covering lab access, biosafety levels, emergency procedures, and dual-use research concerns.
  5. Biosafety and Biosecurity Measures
    • We implement PPE use, inventory controls, physical access controls, data access measures, waste management, and medical surveillance to ensure safety.
  6. Additional Risk Management Actions
    • We have participated in safety training and consulted with experts to refine risk management strategies. Some experimental designs have been modified to enhance safety.

Conclusion

Through a combination of compliance with regulations, expert guidance, thorough training, and proactive risk management, we ensure our project is conducted safely and responsibly. We are dedicated to minimizing risks and maximizing positive outcomes.

Additional Notes

  • Further Improvements
    • We are open to suggestions from the iGEM community on improving our safety practices.

Developing Aptamer-Based Detection Tools for Lung Cancer

In order to diagnose complex diseases like lung cancer more accurately, there is an urgent need for highly sensitive, orthogonal, and broad-spectrum detection tools. These tools must detect various disease biomarkers, including proteins, miRNAs, and small molecules.

Aptamers (APtamers) are artificially screened structured oligonucleotides that fold into highly specific spatial structures via base interactions, allowing them to bind tightly to target biomarkers. The aptamer selection process typically involves the SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique, which selects sequences with the highest affinity from a large library of random sequences.

However, due to the limited research on lung cancer biomarkers and their corresponding aptamers, we chose thrombin, a biomarker with a well-characterized structure and a variety of aptamers, to validate our approach. By selecting two aptamers that bind to different sites on thrombin, we were able to obtain signals of sufficient strength.

Using EMSA, we confirmed that both aptamers bind independently to thrombin. Furthermore, through fluorescence resonance energy transfer (FRET), we demonstrated that these two aptamers, binding to distinct sites, can simultaneously interact with thrombin to form a trimeric complex. Next, we linked the two halves of a cleavage protease to the two aptamers using an unnatural amino acid (this linkage is still under investigation), and showed that the protease can dimerize and exhibit cleavage activity in the presence of SPOC (to be further detailed in future work).

Following validation of the aptamer-thrombin combination, we plan to implement a signal amplification mechanism to detect weaker signals more effectively.