First, we identified several luminescent proteins and transfected cells with expression vectors for these enzymes, including Aequorin, Firefly Luciferase, and Renilla luciferase. Because all of these enzymes were capable of oxidizing coelenterazine, we used them to test and compare the amount of light they could generate when mixed with a given concentration of coelenterazine. To run the experiment, we transfected 293T cells with one of the enzymes, lysed the cells, and mixed the cell lysate (which now contains the enzyme) with a coelenterazine solution. We then placed the samples into a luminometer, which analyzed the light emitted by the solutions to determine which enzyme was the best at producing light. We found that Renilla luciferase was the brightest enzyme, while Aequorin was unfortunately, the least bright. While the other enzymes were could emit more light, Aequorin was the only enzyme suitable for further experiments, and therefore proceeded with the next stage of experimentation with Aequorin.

Our next goal was to more completely characterize Aequorin, as well as our light-activated ion channel, Channelrhodopsin-2 (ChR2) and a mutated, high efficiency version of ChR2, CatCh. To do this, we transfected 293T cells with both Aequorin and either ChR2 or CatCh and grew them in the presence of coelenterazine and Ca2+, which were both required for proper Aequorin function. After irradiation with blue light that should have opened the ChR2 and CatCh ion channels, we analyzed the samples in the iVIS Lumina imaging system. Through the extreme photon-level resolution afforded to us by the IVIS Lumina imaging system, we hoped that we would be able to detect even the tiniest changes in light output, an indication that our cells were receiving and sending signals like we had planned. Unfortunately, our understanding of ChR2 and CatCh function were incomplete, and our experiments showed that ChR2 and CatCh were seemingly unopened by the blue light.

Believing that our ChR2 was not functioning correctly, we used an alternative method to detect the influx of calcium that an open ChR2 or CatCh channel would have caused: calcium sensitive dyes. We treated our cells transfected with only ChR2 with a dye known as X-rhod-1 that glows brightly when in the presence of calcium. We also used a far more intense laser from a confocal microscope that we hoped could force the ChR2 to open. Unfortunately, our results were again, less than spectacular, so we switched to yet another calcium sensitive dye, Fura-2, which also seemed to give us confusing results.

Ultimately, we believe that while the light emitting portion of our light-based cellular signaling project was reasonably successful, due to the our inability to get Channelrhodopsin to work, we were unable to fully realize our dream of a light based cellular signaling system. However, we believe that we have identified the issue. After revisiting the experiment towards the end of our experiments, we found that several papers have implicated all-trans-retinal as an important cofactor in the proper function of ChR2 and CatCh. Therefore, we believe that the lack of retinal in our growth medium was the primary factor behind the failure of our experiments. Unfortunately, due to lack of time, we were unable to repeat the experiments, but we hope to continue our characterization.