Genetically modified organisms escaping from lab is a serious problem since it will bring unpredictable impacts to our ecosystem. In our part design and production line, we were very conscious of biosafety. The yeast will pass the following process to achieve complete destruction. We also dealt cautiously with waste produced from the experiment and participated in safety training to ensure everyone conducting experiments had a minimal probability to cause biohazard pollution.

Filtering system

  The average size of the P. pastoris used is about 4–6 μm (Gmeiner, C. et al. 2015), and the filter we used is 33 kDa, which is equivalent to the size of tens of nanometers (Bacher, G. ey al. 2001), more than sufficient to trap all the yeast.

Heating system

  According to the literature (Martínez, D. et al. 2015), heating at 70 °C for 1 day, and incubate at 30 °C for 48 hr. Under this temperature, it is sufficient to kill any yeasts that escape from the Filtering system. The minimum operating temperature of Extrusion Granulation will be over 110 °C. Therefore, no yeast will escape from the production line we designed.

Following were our heating test:

Figure1: P. pastoris heated at 30 °C for 1 day , and incubate at 30 °C for 48 hr.
(left: 0.005, right: 0.05)

Figure2: P. pastoris heated at 50 °C for 1 day , and incubate at 30 °C for 48 hr.

Figure3: P. pastoris heated at 70 °C for 1 day , and incubate at 30 °C for 48 hr.

  We found that our P. pastoris not survival after were heated at 50 °C for 30 minutes. Thus, our production line heat process would kill P. pastoris passing through the filter.

Reference

Gmeiner, C., Saadati, A., Maresch, D., Krasteva, S., Frank, M., Altmann, F., … Spadiut, O. (2015). Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase. Microbial Cell Factories, 14(1). doi:10.1186/s12934-014-0183-3

Bacher, G., Szymanski, W. W., Kaufman, S. L., Zöllner, P., Blaas, D., & Allmaier, G. (2001). Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses. Journal of Mass Spectrometry, 36(9), 1038–1052.doi:10.1002/jms.208

3. Martínez, D., Menéndez, C., Echemendia, F. M., Hernández, L., Sobrino, A., & Trujillo, L. E. (2015). Kinetics of sucrose hydrolysis by immobilized recombinant Pichia pastoris cells in a batch reactors. J Microb Biochem Technol, 7, 294-6.