Safety

Overview

Fabrevivo is designed as a biological approach for the degradation of textile waste through cellulosome complex by assembling cellulases, PETase, and boosters on a mini-scaffold surface display system on host cells. Our lab design includes using Kluyveromyces marxianus, DH5α, and BL21 as our hosts to produce the designated enzymes and proteins needed. We aim to attain a closed loop by breaking down and recycling the cotton and PET components in textiles and clothing to achieve more efficient use of resources and greener industry.

Project Safety

Wet lab

In our project, the two strains of E.coli that we initially used for the construction of plasmids and expressions of enzymes and proteins were DH5α and BL21, which are common chassis and proved safe for use. Moreover, Kluyveromyces marxianus and Saccharomyces cerevisiae we later selected to use are food-grade yeasts; they function most notably in bakeries, the production of ethanol from food waste streams, and are particularly associated with fermented dairy products such as kefir [1]. The cellulases, boosters, dockerins, and scaffold proteins such as CBHII, NpaBGS, LPMO, and CipA are also non-toxic. During our experiment, we extracted our enzymes through ultrasonication and then by ultracentrifugation. The supernatant and the sediment were separated from the lysate. Finally, we filtered the supernatant to ensure that no microorganisms were left in the needed proteins, which corresponds with the principle of ensuring purity and preventing engineered microbial from leaking into the environment. Afterwards, we assembled our surface display system composed of enzymes and mini-scaffolds in the clean bench to minimize the risk of microbe leakage and leaving the contents exposed to the environment. Our peri dishes and other lab waste were also sterilized and adequately treated before disposal. In the laboratory, areas with different functions were separated clearly to prevent chemical contamination and leakage. For instance, the contaminated area for SDS-page and agarose electrophoresis, areas for general experiments, rooms for writing, classifying data, and conference rooms. Additionally, our team members who participated in the experiments were instructed by professional instructors and teachers in the lab; they supervised the process of handling microbes and using specialized apparatus such as clean benches, providing consistent support and administration.

Lab Safety Precautions

• Wear gloves and goggles when handling microbes and using hazardous chemicals
• Do not wear clothes with loose sleeves when doing experiments to avoid contamination
• Tie back long hair when doing experiments
• Maintain a clean and organized environment when doing experiments
• Remember to turn off the UV light before using the clean bench
• Do experiments that involve hazardous chemicals in well-ventilated areas
• Clearly label substances containing microbes and chemicals used in the lab
• Disinfect lab waste such as pipettes and centrifugal tubes
• Wash used glassware with bleach, detergent and spray with 70% ethanol to remove microbes

Human Practice Safety

• We were fully aware of ethical issues when conducting the public questionnaires. We avoided problems in research that raised concerns about the welfare of participants at all costs. Therefore, we closely followed ethical guidelines in consideration of the welfare of participants and broader society.

1. Informed consent We valued and gave participants informed consent before the questionnaire, explaining the purpose, confidentiality, contact information, and other related aspects of our research. Therefore, participants have full acknowledgement and could voluntarily choose to participate. Of 586 participants, 568 agreed to participate.

   

   figure: our informed consent

2. Right to withdraw As this is an online questionnaire, the participants can drop off the survey at any time without penalty. If participants did not agree to the informed consent form, we kindly ended the questionnaire for them without any pressure.

3. Protection from harm -Participants in a questionnaire have the right to be protected and should not be exposed to greater psychological risk than in daily life. Therefore, we were careful when creating our questions. We believed participants would not feel any embarrassment, fright, or stress after thoughtful consideration.

4. No deception -We constructed the questionnaire without deception and always presented accurate information about our project, aim, and context.

5. Confidentiality -To build trust between us researchers and participants and maintain integrity, we protected participants' data and made them anonymous the whole time. We avoided unauthorized access to their names and location. When we analyzed and presented data, we did not use the names of participants.

6. Privacy

• We valued human ethics when conducting the questionnaire and refrained from asking questions regarding privacy. We only asked about participants' age and gender to get a general sense of the representatives of our sampling method.

7. Debriefing -At the end of the questionnaire, participants can ask questions and share their thoughts and opinions. (see Human Practice: Public Questionnaire, human ethics)

References

[1].Varela, Javier A. et al. “Applications of Kluyveromyces marxianus in Biotechnology.” (2017).