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Download Engineering Red Light-Activated Guanylate Cyclase for Use in
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Controlling Bacteria With Light By: Jesse Hinshaw Special Thanks to INBRE, Mark Gomelsky, Gavin Lawlis, and Stacey Formicheva What is Optogenetics? Optogenetics is the regulation of cellular activities directed by light via genes. Basically, I can make cells do certain things when exposed to red light. Why NIR (Near Infrared) light? Benign Deeply penetrates mammalian tissue High precision: activation when and where you need it. 680-880nm Why Should You Care? Host-Pathogen Studies Pathogens are changing all the time: MRSA or mycobacterium tuberculosis Discovery of new targets for antibiotics or treatments Why Guanylate Cyclase? Guanylate cyclase produce cGMP cGMP is almost undetectable by pathogen NOT present in most pathogens Cyclase Domain How is this enzyme made? Cya2 (guanylate cyclase from Synechosystis) Domain Fusion Via Yeast Recombineering Cya2 Light Sensing Domain Point Mutations in IlaC Adenylate Cyclase Light Sensing Domain Same function, different approaches How can we tell it exists? o Sequencing o DNA gels How do we know they work? Guanylate Phytochrome Cyclase GTP RNA Polymerase cGMP Transcription Activator LacZ-Enzyme LacZ Blue Pigment In a perfect world…… Blue colonies produce a sufficient amount of cGMP White colonies DO NOT produce sufficient cGMP What does that ACTUALLY look like? Do my cyclases work? crpA & IlgC3 crpG & IlgC3 Project Future o Random mutagenesis of point mutation construct o Continued optimization o Move to mice for in vivo analysis Finally, Questions?
