Two prototype genetically encoded Ca2+ indicators



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Calcium ions are important chemical signals for cells. They are especially vital for the function of neurons, which rely on calcium ions to relay messages to subsequent neurons. Visualization of this process allows one to elucidate nervous system function by directly observing the communication of neurons. Thus, the neuroscience community constantly seeks high-performance tools that allow for the visualization of calcium ions. In response to this demand, we developed and analyzed two prototype calcium ion biosensors that can be genetically encoded into model organisms. These novel calcium ion indicators utilize near-infrared fluorescent proteins (NIR FPs), which are beneficial because they rely on low energy light. NIR FP-based biosensors require less harmful conditions for model organisms during imaging and offer deeper tissue penetration compared to other calcium ion biosensors. To construct these calcium ion indicators, we inserted the genes of NIR FPs miRFP720 and miRFP670 into the calmodulin gene, a calcium ion binding domain. Using a vector, E. coli was transformed with this DNA to synthesize the indicator proteins. Finally, the proteins were extracted from the bacteria and analyzed using a fluorescence assay. This project was completed with contributions from Yusuke Nasu and Robert Campbell from the University of Tokyo.