4.1 DNA manipulation
All constructs were assembled using available restriction enzymes and T4
ligase, or Hi-Fi assembly from New England BioLabs (NEB) and other basic
molecular cloning approaches. To construct the light-responsive LLPS
system PhASE#1, we acquired gene sequences of FUSLCD (FUS residues
1-212, NP_004951.1), CRY2 (NP_171935.1), CIB1 (NP_195179.2), and GCN4
(residues 2-34, 1W5I_A) from Pilong Li’s lab at Tsinghua University.
The sequences of the other system, PhASE#2, contained SIM (E3
SUMO-protein ligase PIAS2 isoform X1 residues 505-527, XP_006722634.1)
and SUMO (XP_012635485.1), were synthesized by Ruibiotech directly.
Since we only used CRY2 as a light-responsive element, we added an MBP
tag to avoid its aggregation[49]. Plasmids
(Supplementary Plasmid list) contain genes encoding Rluc (Renilla
luciferase, AGU01696.1), XylE (catechol 2,3-dioxygenase,
WP_011005909.1), fluorescence proteins were acquired from iGEM parts
distribution. All sequence ID could be found on NCBI.