Team:Queens Canada/Experiments
Experiments
All of our protocols can be found on Benchling via the following link:
To construct both our biosensor designs we utilized NEB HiFi DNA Assembely to join together mutliple fragments, incorporating all the domains of our proteins and cloning them directly into the linearized expression vector pET-16b. pET-16b was linearized with XhoI and BamHI.The FRET biosensor was broken into the following g-blocks which were ordered for synthesis from IDT: 1) overlap sequence with pET-16B cloning site – Biobrick Prefix – acGFP1- 11AA linker - 28bp overlaps with Glucocorticoid receptor Ligand binding domain 2) A truncated glucocorticoid receptor ligand binding domain AA521-766 3) 28bp overlaps with glucocorticoid receptor ligand binding domain – mCherry - 4 bp Linker – Biobrick Suffix – 35bp overlaps with PET-16b cloning site The Intein splicing system was broken into the following g-blocks which were ordered for synthesis from IDT: 1) 36bp overlaps with pET-16B overlap cloning site – Biobrick Prefix – Kanamycin Resistance AA1-118- variable linker – 36bp overlaps with receptor Ligand binding domain 2) A truncated glucocorticoid receptor ligand binding domain AA521-766 or a truncated estrogen receptor ligand binding domain AA 303-531 3) 28bp overlaps with receptor ligand binding domain – variable length Linker – Kanamycin Resistance AA120 - 273 - Biobrick Suffix – 36bp overlaps with PET-16b cloning site The NanoLuc luciferase system was constructed through cloning an Anderson Constitutive promoter (Part:BBa_J23100) into the MCS of pNL1.1 (gift of Promega). Upon determination of NanoLuc properties, a RFC10 compatible version with prefix and suffix was ordered for synthesis from IDT. See our experiments