Team:SCAU-China/Safety

MESEG

Safety

Safety is the first priority. SCAU-CHINA always pays attention to safety.



Biosafety

Kill switch


We have also designed a suicide pathway for transgenic cells, which will be verified by experiments later.

Figure 1: Design of kill switch


Since long-wavelength light such as near-infrared light has less attenuation in the environment than short-wavelength light, and plants mainly use red light and blue-violet light, infrared light has little effect on the growth of Chlamydomonas reinhardtii. Based on this principle, we choose near-infrared light as the factor that triggers cell death. In the device, we will use filters to filter out the near-infrared light. Once the algae cells leak into the environment, near-infrared light will induce Bphp1 to transform into the PR state and form heterodimerization with Ppsr2. The nuclear localization signal (NLS) fused to Ppsr2 facilitates the translocation of heterodimers to the nucleus. Next, the TetR fused to Bphp1 interacts with the tetO DNA repeat sequence, and the VP16 fused to Ppsr2 recruits the transcription initiation complex and triggers the transcription of suicide genes (such as ribonuclease). Related regulatory elements have been successfully verified in mammals (Kaberniuk et al., 2016).




Cell fixation and semipermeable membrane


We pay great attention to biosafety. To prevent the leakage of transgene into the environment, we will fix cell using sodium alginate embedding method. Sodium alginate is considered as a good carrier with high activity and mass transfer effect. However, alginate gel has some negative features, such as low mechanical strength, easily to break, and easily to dissolve in solutions containing high concentration of phosphate and cations like as K+, Na+, Mg2+ (Zhang et al., 2013). In recent years, polyvinyl alcohol (PVA) was introduced into sodium alginate, which can enhance the mechanical strength of immobilized pellets (Kamoun et al., 2015). We will use a mixed fixation method with PVA and sodium alginate in this project. In addition, we designed a device in which a cellulose acetate semipermeable membrane was installed. The cellulose acetate semipermeable membrane has a pore size (5 μm) smaller than the cell size of Chlamydomonas reinhardtii (10 μm), and it is non-toxic to cells, so it can be used to prevent cell escape and maintain the relative stability of the internal environment of the device. For more information, see the hardware page.




Organisms


In this project, we used the single-cell green algae--Chlamydomonas reinhardtii CMJ30 strain, which exists in natural water and has no pathogenicity to humans . Relevant tests are strictly standardized in the laboratory to prevent the leakage of engineered Chlamydomonas reinhardtii. We also used yeast strain BY4741 and Escherichia coli DH-5α strain in the project. Experiments were strictly operated in the hood of the laboratory and without any leakage.




Lab safety

In the laboratory, we pay much attention to the use of toxic reagents, such as nucleic acid dyes. We strictly follow the experimental operation rules and no laboratory safety accidents have occurred. The laboratory is equipped with safety equipment for different situations, such as eye and body showers, fire extinguishers, first-aid boxs, and emergency exits.




COVID-19

From May to June 2021, the COVID-19 delta variant virus broke out in Guangzhou, China. Fortunately, under the leadership of the government and the Communist Party of China, we have successfully defeated the virus. During the epidemic, we maintained social distance, insisted on wearing masks, and performed nucleic acid tests to ensure our safety.




References

  1. Kaberniuk, A.A., Shemetov, A.A., and Verkhusha, V.V. (2016). A bacterial phytochrome-based optogenetic system controllable with near-infrared light. NAT METHODS 13, 591-597.
  2. Kamoun, E.A., Kenawy, E.S., Tamer, T.M., El-Meligy, M.A., and Mohy Eldin, M.S. (2015). Poly (vinyl alcohol)-alginate physically crosslinked hydrogel membranes for wound dressing applications: Characterization and bio-evaluation. ARAB J CHEM 8, 38-47.
  3. Zhang C, Wang J, and Yang H, et al.   (2013). Review on immobilized cell technology and its application in water treatment. Technology of Water Treatment 39, 1-4.