The researchers evolved variants of the light-responsive transcription factor El222 that could activate in green light. The discussion focuses on the G80R and T83A variants, whose changes lie near the light-sensing region of the protein. Their behavior illustrates how changes to a protein can alter its response to light without replacing the chromophore, opening possibilities for controlling biological outputs with different colors.
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G80R and T83A are two distinct variants, not a requirement that both mutations occur together in one protein.
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29:47The first thing we're going to do is spectrally modify the light oxygen voltage domains of a certain protein called EL222. This is a protein that is ubiquitous in research. Okay. Um it's a transcription factor. So what that means is this protein I shine light on it that's going to attach to DNA whatever part of DNA that you want. And then that part of DNA is going to be transcribed into mRNA. and it's going to cause the gene to be expressed. So, we can turn on and off genes using EL222. >> I shine blue light of 450 nanometers
30:30>> and whatever gene of interest is going to turn on. >> Interesting. >> That's an incredible technology already. >> Right. >> Okay. >> Right. >> But it's only one color. >> Okay. What if I wanted to turn on two genes? >> Mhm. >> Right. >> Mhm. Well, you could say, well, I just get an EL222 on gene A and a EL222 on gene B. But then when I turn on blue light, both are going to get expressed. >> What if I want to turn on one and then maybe sometime later I want to turn on the on the other and then I want to turn on both later. I'd need two colors of light, >> right? >> So, so currently we sort of have a very naive way to start the factory. Yeah. >> We can basically say start the whole factory. >> Yeah. >> What we're trying to say is we want to
31:11start the chip department only. >> Yeah. And then maybe a time later >> later we'll get the phone department going because the chips are ready or whatever it might be. >> Exactly. So let's get a little bit into this um this protein here EL222. Okay. A lot of these proteins that are light sensitive use something called a chromophore. Okay. This particular one uses a flavin monucleotide chromoplur. That's the FMN. If you look at the protein here, the bulk is the protein and then that little gray sorry no the the bulk of the protein is in gray. the black little compound there that looks like a nucleotide. Nucleotide is the building block of DNA. In this particular case, that little black molecule needs to be there. That black
31:54molecule is going to absorb the blue light photon, go into a higher energy state. Like in quantum mechanics, just classic quantum mechanics, it's going to go into a higher energy state. The protein is then going to sense that higher energy state and change its shape. >> I see. >> Okay. M and for the longest time that blue photon which is at 450 nanome blue remember is high energy >> which means that that chromophore which is the little triggering molecule requires a high energy photon to bump up to that high quantum state and then that causes the shift in the protein structure. Some random alpha helix goes from one state to another state and then that activates the protein. The
32:35challenge is that chromophore is pretty happy with blue light. >> It doesn't want to accept other nanometer wavelengths, right? So shifting the absorption to let's say a lower energy green light cuz green light there's a bunch of lasers we have with green light. It would be really nice if we could make a chromophore that shifts to green light. >> Okay. >> Okay. >> Okay. >> How do we do that? >> Yeah. >> Right. Because we talked about purple lasers recently. >> Yeah. And >> but that's hard, >> right? Green light is easier because it's lower energy, so it's it's fine, right? Right. Um, so I'd like to be able to create an EL222 that doesn't just respond to blue light. But there's another one. There's another version
33:16that responds to green light. That way, if I want to start the chip chip department, I shine the blue light. Then if I want to ch start the phone department, I shine the green light and I have independent control of which gene I'm going to turn on. We we we want to have more granular ability to turn the factory on to generate the proteins we want to under the circumstances we want to and then be able to also stop that process >> when we want to and then have this control of saying if the factory starts producing the thing we don't want it to do the factory will automatically shut down. >> Yeah. Yeah. And so I want like I want I want multiplexing ability. So here's what here's what they did. They they selected under green orange and red
33:57LEDs. And again, you do the same thing. You do directed evolution. You shine it on green. Those that survived make it through. Those that don't. Oops. >> Mhm. >> What they found was key mutations. They actually were able to create this almost impossible light oxygen voltage transcription factor. The chromophore didn't change. Okay. the little black molecule didn't change. >> The original triggering one we talked about >> didn't change. It turns out there's some intermediate state that that thing has that does respond to green light. It goes into an intermediate quantum state, but the original protein was not sensitive to that change
34:39>> through two changes in amino acids. There was at the 83 at the 83 location T went to A. So the theonine went to alanine. One amino acid was switched for another. And at the 80 location, the glycine was moved to an arginine. Both of those 83 and 80, as you can see,
Molecular BiologyProtein EngineeringSynthetic BiologyBioengineering