Genetically encoded REDOX biosensors Future Plan – Culture Platform Matthew Leslie
Outline: Undergrad biosensor work: • Fluorescence • GFP (Green Fluorescence Protein) • REDOX reactions • Single protein sensors VS. FRET (Förster/Fluorescence Resonance Energy Transfer) • 5. Experimental design Future VMSP progect: • Initial design • Future work
Objective: Develop and validate FRET and single protein-based biosensors that allow for real time visualization of intraorganellar redox potentials
Fluorescence Fluorescence is a process in which a molecule absorbs a high-energy photon, and re-emits it as a lower-energy photon with a longer wavelength. Energy = h/λ 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon - the atom is fluorescing.
Green Fluorescent Protein GFP discovered – 1962 , cloned – 1992 Light-stimulated GFP fluorescence is species-independent and does not require any cofactors. Since 1992 GFP has had expansive applications in biomedical research
Oxidation/Reduction The transfer of electrons in oxidation/reduction reactions is central to metabolism in all organisms. Reduction - gain of electrons Oxidation - loss of electrons Glutathione/Glutathione disulfide: Most important intracellular redox pair Made in Cytosol ( 1-10 millimole concentration in cytosol) Glutaredoxin GSSG 2 GSH Glutathione reductase
Sensor design Single protein vs. FRET based approach: Single-protein sensors rely upon the formation of a disulfide bond contorting the internal chromophore of a GFP molecule FRET constructs rely upon a redox-sensitive peptide Placed between pair of fluorescent proteins sharing excitation energy. Total fluorescence vs. FRET efficiency
Endoplasmic Reticulum • Protein folding requires an oxidative environment; • crucial for disulfide bond formation. • The ER must therefor maintain a GSH/GSSG ratio far different from any other cellular compartment • Presents a particular need for a biosensor with less negative reduction potentials. • REDOX control of ER protein • folding machinery has • major implications in disease.
Characterization of REDOX status • Determine dynamic range and REDOX state of intracellular sensor in real time • Fully oxidize and reduce the cellular environment • Reductant – Dithiothreitol (DTT) • Oxidant – Diamide
Initial design: Culture platform designed to modulate oxygen concentration This platform will support initial proliferation studies of LS174T cells in near hypoxic conditions. Future goal: Culture platform mimicking the human gastrointestinal tract. VMSP project
Culture device considerations: • Co-culturing intestinal microbiota alongside mammalian epithelial cells • Mimicking the intestinal ‘biofilm’ in order to study the host/microbe interactions • Main obstacle: maintaining the anaerobic growth conditions needed for microbial colonization while still supporting aerobic mammalian cell growth