Difference between revisions of "Team:NTHU Formosa/Safety"

Biosafety

We designed a user-oriented programmable signaling pathway system that can be triggered by both soluble and insoluble ligands in the extracellular environment. And the. In this project, BioWatcher, the ultimate goal is to use engineered cells with this pathway to recognize biomarkers in the blood stream and express bioluminescence signal for detection. As the engineered cells are aimed to be used in human body, we need to develop a biosafety system that enable the procedure of erasing all the engineered cells implanted into users as users requested or to deal with the allergic reaction and incompatibility.

For biosafety, we designed a two-stage approach to completely disable and destroy the engineered cells when requested. For the first stage, we use auxin-inducible degron system (AID), which can rapidly deplete protein in cells, to disable the engineered cells in no time as an emergency treatment, as the second stage, which takes more time to proceed, results in cell apoptosis to complete the system elimination.

The auxin-based degron system (AID) is a recreation of the SCF-TIR1 complex for target protein degredation in nonplant cells. When auxin is introduced into the system, it promote the interaction of E3 ubiquitin ligase and the degron-targeted protein and results in the degradation of protein targeted by degron. Therefore, Degron is added at the end of the GBP splits. Introducing auxin into the users to degrade split GBP expressing on the engineered cell membrane is the first step to disabled the BioWatcher system. To complete the elimination of the whole system in the users, we involve another set of gene circuit, reverse caspase 3 driven by Auxin promoter, into the engineered cells to cause cell death.

As the users intend to abolish BioWatcher, they can intake auxin to cause both the degradation of split GBP expressing on the engineered cells and the apoptosis of the engineered cells. The protein degradation will only take 9 minutes as an emergency treatment as the cell apoptosis will need 12 to 24 hours to proceed but results in the complete system removal.

Reference：

1. T Ulmasov, Z B Liu, G Hagen, T J Guilfoyle (1995). Composite Structure of Auxin Response Elements
2. Bram G. Lambrus, Tyler C. Moyer, Andrew Holland (2017). Applying the auxin-inducible degradation (AID) system for rapid protein depletion in mammalian cells
3. Kohei Nishimura1, Tatsuo Fukagawa2, Haruhiko Takisawa1, Tatsuo Kakimoto1 & Masato Kanemaki (2009). An auxin-based degron system for the rapid depletion of proteins in nonplant cells
4. Katrin Daniel, Jaroslav Icha, Cindy Horenburg, Doris Muller, Caren Norden, JorgMansfeld (2018). Conditional control of fluorescent protein degradation by an auxin-dependent nanobody
5. Liangyu Zhang, Jordan D. Ward, Ze Cheng, Abby F. Dernburg (2015). The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans
6. Emmanuel Caussinus, Oguz Kanca & Markus Affolter (2011). Fluorescent fusion protein knockout mediated by anti-GFP nanobody