Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Tariq Brown - Central Michigan University
Co-Author(s): Gerard Lambert and Nathan C. Shaner, The Scintillon Instiute, San Diego, CA; Ute Hochgeschwender, Central Michigan University, Michigan, Mt. Pleasant
We developed a technology which uses ‘biological’ light, i.e. light produced by a protein, a luciferase, to activate optogenetic elements upon systemic application of a small-molecule substrate, coelenterazine (CTZ). Depending on the biophysical properties of the optogenetic element, light activation in neurons will result in increase or decrease of neuronal spiking. To move this approach of non-invasive photonic control of neurons closer to potential clinical applications, we need light production with substrate concentrations acceptable for human systemic application. We hypothesized that activation of fused opsins will be more efficient as the light emission of the luciferase increases. One way to increase light output of luciferases is to couple them to fluorescent proteins to take advantage of BRET (bioluminescent resonance energy transfer). We engineered fusion proteins of activating and silencing opsins with a synthetic luciferase coupled to a modified mNeonGreen fluorescent protein. We demonstrated robust expression of this new construct, increased light emission compared to other luciferase variants, and efficient activation of opsins by assessing increased and decreased spiking of cultured neurons in multi electrode arrays.Not Submitted
Funder Acknowledgement(s): NSF Eager Grant Ronald E. McNair Program
Faculty Advisor: Ute Hocgeschwender, firstname.lastname@example.org
Role: I genetically engineered the circular pieces of DNA and nucleofected/ virally transduced neurons used during the research. I conduted electrophysiological recordings in order to determine the impact of the luminopsins on spiking activity.