Team:Queens Canada/Lab
Laboratory
To develop a biosensor, we took two design approaches. At the core of both approaches is a modular Nuclear Receptor Ligand binding domain for binding to an analyte. Immediately flanking the ligand binding domain was two linkers of 3-11 amino acids in length, the selection of appropriate linkers was aided by extensive in-silico modelling, and root-mean-square deviation molecular dynamic modelling. On the ends of each linker was our signal transduction system which acts as our method of producing a measurable outcome once a ligand has bound the receptor.
FRET Approach
For the FRET based approach this system consisted of attaching a Fluorescent Protein with a low peak emission to the N-of the glucocorticoid ligand binding domain, as our FRET donor, and a Fluorescent Protein with a red shifted emission to the C-terminal, as our FRET acceptor. FRET function was validated firstly with a fluorometer, and then quantified with Confocal microscopy.
Intein Approach
For the intein based approach the chosen signal transduction system was a split NanoLuc luciferase which would be joined through splicing together two halves of the RecA split intein. To start however, we first sought to validate our intein splicing in bacterial system as selected through kanamycin resistance, and the utility of NanoLuc luciferase as a strong light source. Upon favorable results with NanoLuc, a portable luminometer was constructed and further tested (see hardware).