In vitro reconstitution of liquid-liquid phase separation in regulating gene transcription

• Main Author: Gan, Jia Yun
• Other Authors: Miao Yansong
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/143324
• Institution: Nanyang Technological University

Liquid-liquid phase separation (LLPS) has become a popular topic of study as more evidence of this process being involved in major processes, such as gene transcription of living cells, are being discovered. In this study, we are interested in determining the conditions where nuclear transcription factor TF9 and TF9-BFP will undergo LLPS in vitro, and the effects of the addition of TF11 and TF11-mcherry on TF9 phase separation. TF9, TF9-BFP, TF11 and TF11-mcherry proteins were extracted from transformed bacteria cultures, but only TF9 was successfully purified. TF9-BFP still had many impurities while TF11 and TF11-mcherry could not be purified during our time of study. Purified TF9 was subsequently incubated under varying polyethylene glycol (PEG) concentrations and Sodium Chloride (NaCl) concentrations size to observe for any formation of droplets. TF9 was seen to start forming droplets at a concentration of 2.5 μM when incubated with 10% PEG and 150 mM NaCl buffer. The NaCl condition required for phase transition could not be determined in this study. Our results imply that TF9 do undergo LLPS under specific conditions and this in turn could shed some light on the microenvironment in the cell that triggers these condensates to form.