Safety

While the application of cucumber transgenics is exciting, the safety of genetically modified (GM) cucumbers needs to be safeguarded through rigorous scientific assessment and regulatory measures. Despite the potential advantages of GM technology, its safety for human health and the environment must be ensured before it can be applied with confidence. Fully aware of this and to ensure the safety of all our team members, our team takes all necessary precautions to ensure that we do not cause any harm to the environment or individual health.

Chassis Safety

We use E. coli DH5α, yeast cell EGY48, and Agrobacterium GV1301 to ensure the safety of our project. These microorganisms are categorized as Risk Group 1; they pose a low risk to human safety and the environment.

Part Safety

For our purpose of controlling the synthesis of flavonoids in cucumber, we introduced two genes, CsPAL and CsTBH. Before we were to use these two genes, we checked the origin of the genes, both genes are from the cucumber genome and both belong to the white list provided by iGEM.

Results

Once we constructed the plasmids, we transformed them into E. coli DH5α and then cultured them on resistant LB medium and performed colony PCR on the single cultures.Here are the colony PCR results.

Fig.1 Results of colony PCR

Once our gene sequencing was correct, we began our yeast single heterozygote experiment by transferring the yeast cells to SD plates for culture. Then, the yeast cells were transferred to the SD plate and cultured on X-Gal chromogenic medium for color development. The whole process did not involve any serious safety issues, and at the end of the experiment, we sprayed the experimental area with alcohol to ensure that the residual yeast cells would be killed to ensure the safety of our laboratory environment.

Fig.2 Yeast single hybrid results show that CsTBH can bind the promoter of CsPAL

Laboratory Safety

Prior to starting the lab, our advisor spent a great deal of time giving us a detailed lab safety course and guiding us through the basic lab procedures. To ensure the safety of our team members in the lab, we conducted all of our experiments under the supervision of at least one lab instructor. We also had a special hazardous area in the lab where we performed gel electrophoresis and added nucleic acid dyes. We were strictly prohibited from bringing food into the lab and always wore appropriate clothing, lab coats, and gloves for our experiments. At the end of the experiment, we cleaned up everything and stored the waste accordingly. In addition, we participated in fire drills to further ensure the safety of everyone in the lab.

References:

[1] Zhou J, Liu G, Zhao Y, Zhang R, Tang X, Li L, Jia X, Guo Y, Wu Y, Han Y, Bao Y, He Y, Han Q, Yang H, Zheng X, Qi Y, Zhang T, Zhang Y. An efficient CRISPR-Cas12a promoter editing system for crop improvement. Nat Plants. 2023 Apr;9(4):588-604. doi: 10.1038/s41477-023-01384-2. Epub 2023 Apr 6. PMID: 37024659.

[2] Kleter GA, Kuiper HA, Kok EJ. Gene-Edited Crops: Towards a Harmonized Safety Assessment. Trends Biotechnol. 2019 May;37(5):443-447. doi: 10.1016/j.tibtech.2018.11.014. Epub 2019 Jan 4. PMID: 30616999.

[2] Weeks DP. Gene Editing in Polyploid Crops: Wheat, Camelina, Canola, Potato, Cotton, Peanut, Sugar Cane, and Citrus. Prog Mol Biol Transl Sci. 2017;149:65-80. doi: 10.1016/bs.pmbts.2017.05.002. Epub 2017 Jun 16. PMID: 28712501.