by Jingren Carl Gu
This Summer, under FUTI’s generous support, I spent 7 weeks at the Bito Lab in the UTokyo School of Medicine. I took part in an ongoing project by Dr. Hajime Fujii to design a genetically encoded calcium indicator (GECI). A GECI is a calcium sensitive fluorescent protein that scientists introduce into the brain. When a neuron fires, a flux of calcium enters the cell through voltage-gated calcium channels, and the GECI lights up, signalling to the observer that the neuron was just active. However, not every GECI is the same. Some are like distant stars in a night sky: their lights are dim, but occasionally, they brighten up 10 folds and blink at you under the dark canvas of the horizon. Some are like the full moon: its gentle light showers over the vast terrain, beholding the night with an everlasting glare. Starlight is weak — it cannot be seen clearly like that of the moon. Moonlight is static—- it cannot blink like that of a star. That is the conundrum: scientists need a molecular indicator that is both bright, so they easily stand out under the noisy background of the brain, and shiny, so that when the cell becomes active, there is a dramatic change in brightness that can be easily measured. In other words, Dr. Fujii and I were looking for a GECI that had both brightness and dynamic range –– we were looking for a moon that blinks.
The procedure, on the other hand, was much more down to earth. We cultured cells that were randomly expressing GECIs with different mutations and tested their responsiveness. These mutations, through a myriad of molecular interactions, gave each version of the GECI a unique brightness and dynamic range. We looked for precise combinations of mutations that would result in a calcium indicator that had both high brightness and high dynamic range.
This was not an easy feat, however, as there were hundreds of thousands of potential combinations! Through each screening, we incubated several hundreds of cell cultures, which expressed several hundred versions of the GECI. We used these as a representative set of the entire set of possibilities. We screened for their brightness and dynamic range and examined the copies that produced the best performance. We then picked out mutations that potentially led to their high performance and fixed these mutations onto the next generation of fluorescent probes, hoping to improve the population’s overall quality in the subsequent round. By the end of the 7 weeks, we have undergone several rounds of screening and examined thousands of candidate GECI mutants. From them, we identified two families of GECIs that consistently met our standards and a few specific variants that had both outstanding brightness and dynamic range.
Outside of the lab, I was an avid ramen enthusiast. For countless evenings, after a long day of work, I would stand in front of the ramen ticket machine, my mouth watering as the coins fell into the coin slot and the menu items lit up. I also went on many dinner gatherings with my lab mates, through which I learned about where locals go for fulfilling and cheap meals, what life in research is like, and how to navigate Shinjuku’s busy alleyways.
One night, when I was walking down the streets of Akihabara, I saw a row of Gashapon machines lined up against the road. To play, you simply insert a coin and turn the handle. After a crackling sound, the machine returns a capsule that contains a random toy inside. Since there are many varieties, whether you get the toy you want is completely up to chance. I thought this resembled my research project, as the mutations in the GECIs were also random, and it took luck to stumble upon an effective mutation out of thousands of possibilities. After hearing this thought, Dr. Fujii objected: “Although luck is involved in each round of screening, after each round, we always look for patterns in the data and use them to improve our odds in the next round. No matter what the result is, our knowledge always grows.” That is what I took away from this experience. You must work diligently and meticulously for a long time to earn a chance to test your luck. Then, no matter if you are lucky or not, you will have learned something, and you will use that knowledge to improve your chances in the next try. Eventually, your chances will accumulate, so much so that when you peek into the microscope, you will really see a moon that blinks.
