Our laboratory experiments were conducted perfectly in tandem with our hardware, and software efforts as well. Therefore, we began construction of our Biosensors simultaneous with building our pacifier, and calibrating our luminometer.
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 - overlaps with the Glucocorticoid receptor Ligand binding domain
2) glucocorticoid receptor ligand binding domain
3) overlap sequence with glucocorticoid receptor ligand binding domain – mCherry - 4AA Linker – Biobrick Suffix – 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) Overlaps with pET-16B overlap cloning site – Biobrick Prefix – Kanamycin Resistance AA1-118- variable linker – 36bp overlaps with receptor Ligand binding domain
2) glucocorticoid receptor ligand binding domain or estrogen receptor ligand binding domain
3) overlaps with receptor ligand binding domain – variable length Linker – Kanamycin Resistance AA120 - 273 - Biobrick Suffix – Overlaps with PET-16b cloning site
To calibrate our luminometer, we sought to use the very bright NanoLuc Luciferase as the source of luminescence.
The NanoLuc luciferase expression system was constructed through obtaining a Nanoluc containing vector from Promega, then cloning an Anderson Constitutive promoter (Part:BBa_J23100) into the vector for bacterial expression. Upon determination of NanoLuc properties, a RFC10 compatible version with prefix and suffix was ordered for synthesis from IDT and used for subsequent testing.
Please find many of the protocols we followed below:
Diagnostic Restriction Digest ELISAs Error Prone PCR gBlock Gibson Assembly Glo Lysis for Luciferase Glycerol Stocks His-Tag Nickel Chromatography Kanamycin and cortisol agar plate prep
LB Broth and Agar Plates
Liquid Culture Lysis by Sonication MiniPrep
Resuspension of gBlocks Gene Fragment Streaking Plates
