Development of a modular dual vector system via golden gate cloning to assay E3 ligase activity in plant protoplasts

Identifier:  TFG:8980

Handle: https://hdl.handle.net/20.500.11797/TFG8980

Authors:  Arjona Martí, Aida

Abstract:
Multiple protein homeostasis networks control proteins from synthesis to degradation. Protein abundance depends on production and degradation rates. Ubiquitin modification determines cellular fate, including DNA repair, cell-cycle regulation, or proteasome-mediated degradation. Assessing the effects of ubiquitin modification by E3 ligases is challenging, as in vivo monitoring often relies on reporter systems. To address this, we developed a dual vector system for ratiometric analysis of E3 ligase activity in plant protoplasts. Constructs were generated using Golden Gate cloning, assembled into transcriptional units, and verified by sequencing. We produced seven sensors and two effectors, enabling precise monitoring of protein stability.