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Safety

Describe all the safety issues of your project.


 Lab Safety

    This project has a biosafety level of 2. To manage the risks associated with this project, safety protocols were followed, and all procedures were planned and approved by the Chulalongkorn Lab advisors to ensure adherence to biosafety standards. Our team received proper training on decontamination, biological waste management, the use of personal protective equipment (PPE), the safe handling of biological spills, and the transportation of biological materials. This training was conducted by lab experts at Chulalongkorn University, ensuring the team upheld high biosafety standards. The lab provided all necessary PPE, biosafety supplies, and guidance to maintain a safe environment throughout the project [1-7].

    When working with Agrobacterium tumefaciens to agroinfiltrate Nicotiana benthamiana, we used a biosafety cabinet to prevent potential exposure through inhalation or skin contact. Safety glasses and lab coats were worn during the infiltration process to prevent accidental exposure to Agrobacterium tumefaciens.

    Additionally, all team members adhered to strict lab protocols by not bringing any external substances, such as food, into the lab and by ensuring that no experimental materials were taken outside the lab. The lab environment was shared with other Chulalongkorn biologists, who, along with our team, strictly follow biosafety regulations to ensure safety at biosafety level 2 [1-7]. Biological waste was properly managed by autoclaving materials as needed to prevent contamination and spread. All used gloves and equipment were disposed of in designated biological waste bins.


 Kill Switch

    We also thought of possible safety systems to prevent the spread of this plant should the transformation develop beyond proof of concept. One of these was a kill switch which would prevent the growth of the plant when exposed to red light.

killsiwtch.png

Fig.3: Vector diagram for the kill switch

The key elements of this kill switch are:

  • Pfr-promoter - This promoter is light inducible and responds to red wavelengths of light so our plant is dependent on a red-light filtered growing environment.
  • Barnase - Barnase is a ribonuclease which causes degradation of single-stranded RNA. We chose this for our killing mechanism because expression of this protein without its inhibitor, Barstar, would result in cell death and thus cause the target plant to die.
  • Terminator Sequence - The Nos Terminator is commonly used in plasmids for genes that are introduced into plants, so we thought it would be a good fit for our purpose.
  • pEAQ-HT-DEST1 - We chose pEAQ for our plasmid background because it is commonly utilized for the transformation of plant cells in Agrobacterium-mediated transformation. The pEAQ-HT-DEST1 plasmid backbone includes:
    • P19 Silencer to reduce the possibility of host plant cells silencing the transfer complex.
    • Marker Genes like Kanamycin resistance for selection of transformed Agrobacterium.

    The advantages of this kill switch are that it is easy to reproduce, with red light being present everywhere in uncontrolled environments, so it would be activated almost immediately if the plant escapes containment without needing any special equipment for activation.

    Unfortunately, this composite part was not able to be tested in the lab environment. If this project were to be developed further, future prospects would include testing of this kill switch to see if it is a viable part.


 References

    1. Chuanchuen R. Biological Spills - Response and Clean Up. Cuvetamr.vet.chula.ac.th. September 16, 2020. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421083831.pdf.

    2. Chuanchuen R. Hands on Practice - PPE. Cuvetamr.vet.chula.ac.th. September 16, 2020. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421082952.pdf.

    3. Chuanchuen R. Decontamination. Cuvetamr.vet.chula.ac.th. September 16, 2020. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421082830.pdf.

    4. Biosafety Equipment — PPE and BSCs. Cuvetamr.vet.chula.ac.th. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_Biosafety%20equipment_PPE%20BSC.pdf.

    5. Biosafety Equipment - Decontamination & Disinfection. Cuvetamr.vet.chula.ac.th. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421075056.pdf.

    6. Chuanchuen R. Biological Waste Management. Cuvetamr.vet.chula.ac.th. September 16, 2020. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421075030.pdf.

    7. Puangseree J. Transportation of Biological materials. Cuvetamr.vet.chula.ac.th. September 16, 2020. Accessed June 13, 2023. http://www.cuvetamr.vet.chula.ac.th/media/content/file/2021/04/CONTENT_FILE_35_20210421081256.pdf.