1 00:00:24,440 --> 00:00:28,028 PROFESSOR: OK, all right, so just very briefly, 2 00:00:28,028 --> 00:00:30,070 I just wanted to remind you because it goes along 3 00:00:30,070 --> 00:00:33,730 with the video, bioluminescence is 4 00:00:33,730 --> 00:00:37,360 light emitted based on a biochemical reaction. 5 00:00:37,360 --> 00:00:41,680 The most common one is the enzyme luciferase, 6 00:00:41,680 --> 00:00:45,520 which reacts with a molecule called luciferin 7 00:00:45,520 --> 00:00:50,690 to undergo a biochemical transformation that uses ATP. 8 00:00:50,690 --> 00:00:52,630 And as a result of that transformation, 9 00:00:52,630 --> 00:00:54,290 there is light emitted. 10 00:00:54,290 --> 00:00:57,190 So you don't have to shine light onto these. 11 00:00:57,190 --> 00:01:00,520 The light comes from the biochemical reaction. 12 00:01:00,520 --> 00:01:04,269 So the assays and a lot of biological work 13 00:01:04,269 --> 00:01:08,170 is based on the luciferin, luciferase reaction, 14 00:01:08,170 --> 00:01:12,580 so the mushrooms that do this will have both luciferase 15 00:01:12,580 --> 00:01:14,230 and luciferin. 16 00:01:14,230 --> 00:01:19,150 So in an organism, you can transfect-- or in cells, 17 00:01:19,150 --> 00:01:22,720 you can transfect cells with luciferase, 18 00:01:22,720 --> 00:01:25,120 and then when you're ready to do the-- 19 00:01:25,120 --> 00:01:28,510 to see the luminescence, the bioluminescence, 20 00:01:28,510 --> 00:01:31,750 you can add luciferin exogenously, 21 00:01:31,750 --> 00:01:33,520 and there's a number of chemists that 22 00:01:33,520 --> 00:01:36,010 are trying to make modified systems that 23 00:01:36,010 --> 00:01:38,890 have different light energies and so on. 24 00:01:38,890 --> 00:01:41,830 So there's a lot of protein engineering 25 00:01:41,830 --> 00:01:46,600 that's taking place based on the luciferin/luciferase pair. 26 00:01:46,600 --> 00:01:51,210 There's all kinds of cool biochemistry going on there, 27 00:01:51,210 --> 00:01:51,880 OK? 28 00:01:51,880 --> 00:01:54,880 Anyone who comes in could come and grab a donut. 29 00:01:54,880 --> 00:01:57,715 We can move them, or do you want a donut? 30 00:01:57,715 --> 00:01:58,670 AUDIENCE: [INAUDIBLE] 31 00:01:59,170 --> 00:02:01,840 PROFESSOR: OK, all right. 32 00:02:01,840 --> 00:02:04,930 I didn't mean to call attention to-- 33 00:02:04,930 --> 00:02:10,690 OK, I want to go briefly back to arrays, DNA arrays, 34 00:02:10,690 --> 00:02:14,620 because I want to explain to you the power of this technology. 35 00:02:14,620 --> 00:02:20,140 So last time, we were talking about arrays which are 36 00:02:20,140 --> 00:02:24,910 basically printed layouts, and I'm-- this is going to be 37 00:02:24,910 --> 00:02:29,420 a very tiny array with six sites on the array. 38 00:02:29,420 --> 00:02:31,180 So it's a two by three array. 39 00:02:34,490 --> 00:02:38,600 The ones that are used in biology 40 00:02:38,600 --> 00:02:40,370 are way larger than that. 41 00:02:40,370 --> 00:02:43,100 You can see those up here-- 42 00:02:43,100 --> 00:02:48,310 far, far more spots, and this is just the size of a glass slide 43 00:02:48,310 --> 00:02:49,850 and at each of these sites, there 44 00:02:49,850 --> 00:02:52,130 would be different DNA sequences. 45 00:02:59,150 --> 00:03:02,420 So they're specific DNA sequences, 46 00:03:02,420 --> 00:03:06,050 and they are printed onto the array with-- 47 00:03:06,050 --> 00:03:08,090 it was originally done just with printers. 48 00:03:08,090 --> 00:03:12,050 Now there's more technological ways of handling this, 49 00:03:12,050 --> 00:03:19,950 and they're usually on silicon or glass slides. 50 00:03:19,950 --> 00:03:23,450 So there's a lot of engineering went into the original arrays 51 00:03:23,450 --> 00:03:27,860 to make these really packed tight distribution of DNAs. 52 00:03:27,860 --> 00:03:30,230 And these would be what are called addressed arrays. 53 00:03:30,230 --> 00:03:32,900 We know what's at each of the sites. 54 00:03:32,900 --> 00:03:35,600 We're aware of the sequences at each of the sites, 55 00:03:35,600 --> 00:03:37,490 and there's really cool chemistry 56 00:03:37,490 --> 00:03:41,130 that would have enabled the disposition of these arrays. 57 00:03:41,130 --> 00:03:43,220 So what these arrays are used for 58 00:03:43,220 --> 00:03:46,980 is to probe complementary pieces of DNA. 59 00:03:46,980 --> 00:03:51,410 So let's say we've got a GHIJ that 60 00:03:51,410 --> 00:03:57,170 come potentially from genomic DNA, and they complement-- 61 00:03:57,170 --> 00:03:59,900 each of them complement one of the sites on the J-K-- 62 00:04:03,440 --> 00:04:06,110 complement one of the sites on the array, 63 00:04:06,110 --> 00:04:08,180 but the DNA that you're screening 64 00:04:08,180 --> 00:04:12,330 is specifically labeled with a fluorophore, OK? 65 00:04:12,330 --> 00:04:17,329 So these would be fluorophore labeled, 66 00:04:17,329 --> 00:04:22,270 and arrays enable you to do is to rapidly screen 67 00:04:22,270 --> 00:04:26,080 either genomic DNA or, actually, much more usefully, 68 00:04:26,080 --> 00:04:30,190 as you'll know now, the transcriptome, so the messenger 69 00:04:30,190 --> 00:04:34,420 RNAs, and I'm going to remind you about technology 70 00:04:34,420 --> 00:04:37,600 to take the messenger RNAs from cells 71 00:04:37,600 --> 00:04:39,730 and then make them an appropriate copy 72 00:04:39,730 --> 00:04:43,220 of a complementary DNA to what's on the arrays. 73 00:04:43,220 --> 00:04:45,190 So let's go take a look at the next slide which 74 00:04:45,190 --> 00:04:47,330 describes the experiment. 75 00:04:47,330 --> 00:04:51,100 So let's say you want to screen populations of cells 76 00:04:51,100 --> 00:04:53,860 where one set of cells is cancerous 77 00:04:53,860 --> 00:04:55,690 and the other one is healthy. 78 00:04:55,690 --> 00:04:58,630 You would collect the lysate from those cells. 79 00:04:58,630 --> 00:05:02,710 You would collect the messenger RNA presumably 80 00:05:02,710 --> 00:05:06,280 based on a tag or a feature of the messenger RNA 81 00:05:06,280 --> 00:05:08,080 that's common to just the messenger. 82 00:05:08,080 --> 00:05:10,580 What would that be? 83 00:05:10,580 --> 00:05:13,660 What would be common to the mature messenger RNA that 84 00:05:13,660 --> 00:05:17,530 would allow you to capture just the messenger RNA, 85 00:05:17,530 --> 00:05:20,140 not all the other RNAs, not DNA. 86 00:05:20,140 --> 00:05:20,730 Anyone? 87 00:05:20,730 --> 00:05:21,230 Yeah? 88 00:05:21,230 --> 00:05:22,202 AUDIENCE: [INAUDIBLE] 89 00:05:22,202 --> 00:05:23,710 PROFESSOR: The poly-A tail. 90 00:05:23,710 --> 00:05:26,080 So you can, with an appropriate resin, 91 00:05:26,080 --> 00:05:28,720 for example, a poly-T support, you 92 00:05:28,720 --> 00:05:32,770 can capture that all, and then in each case, 93 00:05:32,770 --> 00:05:36,010 you would take the messenger RNA collection, 94 00:05:36,010 --> 00:05:38,080 and then what you need to do is turn it 95 00:05:38,080 --> 00:05:42,040 into a complementary DNA sequence. 96 00:05:42,040 --> 00:05:44,830 We're much happier screening at the DNA level 97 00:05:44,830 --> 00:05:47,390 because the RNA is less stable. 98 00:05:47,390 --> 00:05:50,410 So it's really handy to do that conversion, 99 00:05:50,410 --> 00:05:54,640 and you use the viral enzyme, reverse transcriptase. 100 00:05:54,640 --> 00:05:58,450 That's an enzyme that comes from the retroviruses that 101 00:05:58,450 --> 00:06:02,110 are exemplified by the HIV retrovirus 102 00:06:02,110 --> 00:06:04,630 that we'll be talking about in the last week of class. 103 00:06:04,630 --> 00:06:08,560 So you'll learn where reverse transcriptase fits in the life 104 00:06:08,560 --> 00:06:10,780 cycle of the HIV virus. 105 00:06:10,780 --> 00:06:14,320 Then once you've collected the DNA that's 106 00:06:14,320 --> 00:06:17,395 complementary to the RNA from each type of cell, 107 00:06:17,395 --> 00:06:20,110 you label them uniquely, for example, 108 00:06:20,110 --> 00:06:23,200 with green fluorophores or red fluorophores. 109 00:06:23,200 --> 00:06:26,500 It's kind of a global reaction you just paste-- 110 00:06:26,500 --> 00:06:30,850 chemically link the fluorophore to each set of DNA. 111 00:06:30,850 --> 00:06:33,750 And then you basically combine everything 112 00:06:33,750 --> 00:06:37,360 and take a look at how the chips, how the arrays, 113 00:06:37,360 --> 00:06:38,440 light up. 114 00:06:38,440 --> 00:06:41,560 So your typical experiment would be everywhere 115 00:06:41,560 --> 00:06:44,470 you've got something that has a red fluorophore might 116 00:06:44,470 --> 00:06:46,510 bind to a unique site. 117 00:06:46,510 --> 00:06:48,940 Everything with a green fluorophore 118 00:06:48,940 --> 00:06:51,940 might bind to another unique site on the array. 119 00:06:51,940 --> 00:06:57,580 And then if both types of DNA binding to the same site, 120 00:06:57,580 --> 00:06:59,680 that would show up as yellow. 121 00:06:59,680 --> 00:07:03,700 If nobody is binding, it would show up as a blank spot. 122 00:07:03,700 --> 00:07:07,120 So you get these collections where, pretty easily, you 123 00:07:07,120 --> 00:07:10,480 can look at thousands of sequences 124 00:07:10,480 --> 00:07:12,580 to see whether they're complementary. 125 00:07:12,580 --> 00:07:14,270 That's on your array. 126 00:07:14,270 --> 00:07:16,900 Everywhere you see yellow, the cancer cell 127 00:07:16,900 --> 00:07:20,740 and the healthy cell, a binding kind of equally, 128 00:07:20,740 --> 00:07:23,350 so there's nothing disease related there. 129 00:07:23,350 --> 00:07:26,380 So that's an interesting feature because you can say, 130 00:07:26,380 --> 00:07:30,280 well, none of these are problematic segments, 131 00:07:30,280 --> 00:07:34,600 but then you see very clearly spots that are clearly red. 132 00:07:34,600 --> 00:07:40,120 So the sequences of the DNA on the glass or silicon chip 133 00:07:40,120 --> 00:07:42,610 are complementary to sequences that 134 00:07:42,610 --> 00:07:47,020 are unique to the mRNA in the cancer genome, 135 00:07:47,020 --> 00:07:51,130 in the cancer cell line from that lyaste. 136 00:07:51,130 --> 00:07:52,883 So it's pretty cool. 137 00:07:52,883 --> 00:07:54,800 I don't know if any of you have hit that link, 138 00:07:54,800 --> 00:07:57,940 but it's basically this guy is sitting at a bench 139 00:07:57,940 --> 00:08:02,320 and doing an experiment where he's able to do thousands-- 140 00:08:02,320 --> 00:08:05,710 the equivalent of thousands and thousands of experiments just 141 00:08:05,710 --> 00:08:09,370 by using one gene chip to do the probing. 142 00:08:09,370 --> 00:08:14,770 In other cases, you will see spots that are green, 143 00:08:14,770 --> 00:08:18,710 which mean they come exclusively from the healthy cells. 144 00:08:18,710 --> 00:08:22,100 So those would be really the ones you want to interrogate, 145 00:08:22,100 --> 00:08:24,050 and where it's black, nothing's binding, 146 00:08:24,050 --> 00:08:25,390 so it's less important. 147 00:08:25,390 --> 00:08:29,170 So it allows you to go from a grid of thousands and thousands 148 00:08:29,170 --> 00:08:32,710 of different sequences to pick out suspects 149 00:08:32,710 --> 00:08:35,064 in a disease-related situation. 150 00:08:35,064 --> 00:08:36,580 Does that makes sense? 151 00:08:36,580 --> 00:08:38,559 Pretty cool technology. 152 00:08:38,559 --> 00:08:41,030 The original chips were called-- 153 00:08:41,030 --> 00:08:45,340 they were from, I think, Affymetrix, the Affy chips, 154 00:08:45,340 --> 00:08:50,040 and they are really widely used even to this day, 155 00:08:50,040 --> 00:08:52,480 and there are different types of variations, 156 00:08:52,480 --> 00:08:54,430 but I think one of the most useful 157 00:08:54,430 --> 00:08:57,160 and ones that will remind you of certain things we've talked 158 00:08:57,160 --> 00:09:00,070 about in the class is that you can 159 00:09:00,070 --> 00:09:05,500 grow cells of different sort of, you know, histories. 160 00:09:05,500 --> 00:09:07,780 For example, you collect the messenger 161 00:09:07,780 --> 00:09:10,090 RNA because, remember, the transcriptome is 162 00:09:10,090 --> 00:09:13,720 much more interesting to us than the genome. 163 00:09:13,720 --> 00:09:15,590 The chips would have to be a lot bigger 164 00:09:15,590 --> 00:09:19,140 to scan all of the genome, and then you 165 00:09:19,140 --> 00:09:23,100 use reverse transcriptase to make a complementary DNA 166 00:09:23,100 --> 00:09:26,730 of the RNA because it's more stable, more tractable 167 00:09:26,730 --> 00:09:30,360 to work with, and then you put on a fluorophore label, 168 00:09:30,360 --> 00:09:32,700 mix everything together, and see what you get. 169 00:09:32,700 --> 00:09:36,390 So those are my candidate spots. 170 00:09:36,390 --> 00:09:40,410 So if you had an experiment, let me think-- 171 00:09:40,410 --> 00:09:43,780 walk you through what the arrays can do, 172 00:09:43,780 --> 00:09:46,950 but let's make sure we know what they can't do because it's 173 00:09:46,950 --> 00:09:50,080 always important whenever someone says, 174 00:09:50,080 --> 00:09:51,690 I got this technology. 175 00:09:51,690 --> 00:09:54,840 It's going to solve all the problems of the world. 176 00:09:54,840 --> 00:09:57,207 Throw away or your beakers and test tubes. 177 00:09:57,207 --> 00:09:58,790 You just don't need to do any of that, 178 00:09:58,790 --> 00:10:01,720 and you can solve everything with arrays. 179 00:10:01,720 --> 00:10:05,040 So let's say you've got a situation that, in some cases, 180 00:10:05,040 --> 00:10:07,650 although genes may be defective, the messenger 181 00:10:07,650 --> 00:10:09,690 is still produced, OK? 182 00:10:09,690 --> 00:10:14,260 So there's something wrong, but you make the messenger RNA. 183 00:10:14,260 --> 00:10:19,800 So it would still kind of show up normally on the chip, 184 00:10:19,800 --> 00:10:24,810 but the gene defect prevents translation into proteins. 185 00:10:24,810 --> 00:10:27,630 So you get the gene, but you can't translate it, 186 00:10:27,630 --> 00:10:30,540 so it's going to look like a normal gene, 187 00:10:30,540 --> 00:10:33,600 but it's in the translation from the track-- 188 00:10:33,600 --> 00:10:37,690 from the messenger RNA to the protein where things go wrong. 189 00:10:37,690 --> 00:10:40,950 What are you going to see on the array? 190 00:10:40,950 --> 00:10:42,150 Is it going to look healthy? 191 00:10:42,150 --> 00:10:43,710 Is it going to look not healthy? 192 00:10:46,781 --> 00:10:48,420 What's going to be the outcome? 193 00:10:48,420 --> 00:10:53,530 Could I use an array, a DNA array, for this experiment? 194 00:10:53,530 --> 00:10:54,270 Yeah? 195 00:10:54,270 --> 00:10:55,334 What do you think? 196 00:10:55,334 --> 00:10:56,262 AUDIENCE: [INAUDIBLE] 197 00:10:57,190 --> 00:10:58,590 PROFESSOR: Yeah, Yeah. 198 00:10:58,590 --> 00:11:02,460 It's just going to look like everything binds to the chip. 199 00:11:02,460 --> 00:11:04,900 It's not going to be informative. 200 00:11:04,900 --> 00:11:08,700 So in this case, you need to do very different experiments that 201 00:11:08,700 --> 00:11:10,950 are at the protein level, all right? 202 00:11:10,950 --> 00:11:12,810 So I want you to remember that these 203 00:11:12,810 --> 00:11:18,180 are very good to get the genomic sequence, the messenger RNA 204 00:11:18,180 --> 00:11:19,120 sequence. 205 00:11:19,120 --> 00:11:23,490 But if you've got, for example, a protein that is made, 206 00:11:23,490 --> 00:11:25,750 and there's a problem with, let's say, 207 00:11:25,750 --> 00:11:28,020 a phosphorylation by a kinase that 208 00:11:28,020 --> 00:11:30,630 may be critical for function in a cell, 209 00:11:30,630 --> 00:11:33,300 you will not see that in the array. 210 00:11:33,300 --> 00:11:34,530 That will not be visible. 211 00:11:34,530 --> 00:11:37,890 You'd have to do different types of experiments, 212 00:11:37,890 --> 00:11:39,510 for example, with phosphoproteins, 213 00:11:39,510 --> 00:11:42,090 specific antibodies to tell that there was 214 00:11:42,090 --> 00:11:44,310 a disorder in those systems. 215 00:11:44,310 --> 00:11:46,110 So always when someone tells you, 216 00:11:46,110 --> 00:11:49,500 I've got this great experiment, you say, what can it do? 217 00:11:49,500 --> 00:11:52,050 That is awesome, but what can't it do? 218 00:11:52,050 --> 00:11:55,070 OK, I need to worry about that, all right? 219 00:11:55,070 --> 00:12:00,750 OK, so what we've seen so far is we've 220 00:12:00,750 --> 00:12:03,900 seen a variety of fluorescent tools, 221 00:12:03,900 --> 00:12:06,360 for example, to label the nucleus, 222 00:12:06,360 --> 00:12:10,950 either the original ethidium bromide types of stains that 223 00:12:10,950 --> 00:12:15,690 will intercalate into DNA and give you a fluorescent signal 224 00:12:15,690 --> 00:12:17,550 that could be seen on a gel. 225 00:12:17,550 --> 00:12:23,640 We've seen the evolution of ethidium bromide 226 00:12:23,640 --> 00:12:27,695 to less toxic materials, for example, the DAPI stain. 227 00:12:30,420 --> 00:12:34,160 So ethidium bromide intercalates-- 228 00:12:37,420 --> 00:12:40,005 ethidium-- sorry, DAPI binds in the minor groove. 229 00:12:46,170 --> 00:12:49,110 And we saw images of that last time. 230 00:12:49,110 --> 00:12:51,090 One important distinction that is 231 00:12:51,090 --> 00:12:54,150 made with these kinds of dyes is that DAPI 232 00:12:54,150 --> 00:13:04,550 is what's known as a supravital dye, which 233 00:13:04,550 --> 00:13:08,450 means you can use that dye and observe live cells. 234 00:13:08,450 --> 00:13:12,530 So supravital is associated with the types of experiments 235 00:13:12,530 --> 00:13:16,370 that you can do observing cells that are still alive. 236 00:13:16,370 --> 00:13:19,410 The ethidium bromide is not such a dye, 237 00:13:19,410 --> 00:13:26,330 and neither are the antibodies because so ethidium bromide is 238 00:13:26,330 --> 00:13:31,400 toxic, so even though the ethidium bromide gets 239 00:13:31,400 --> 00:13:32,930 into cells, it actually should have 240 00:13:32,930 --> 00:13:38,690 the H here because that would be ethyl bromide in my language. 241 00:13:38,690 --> 00:13:42,080 So ethidium bromide has the H there. 242 00:13:42,080 --> 00:13:43,190 You can't use that. 243 00:13:43,190 --> 00:13:46,580 It's not a supravital dye because it's toxic to cells 244 00:13:46,580 --> 00:13:50,630 because intercalates in the DNA and disrupts replication, 245 00:13:50,630 --> 00:13:54,410 so that is not supravital, so DAPI is. 246 00:13:54,410 --> 00:14:02,000 This guy isn't, and antibodies, which 247 00:14:02,000 --> 00:14:04,550 we learned a little bit about last time, these 248 00:14:04,550 --> 00:14:07,430 are from my description, are simply 249 00:14:07,430 --> 00:14:13,130 reagents to recognize components of biological systems, most 250 00:14:13,130 --> 00:14:17,900 commonly, proteins, and now we're 251 00:14:17,900 --> 00:14:19,640 getting better at making antibodies 252 00:14:19,640 --> 00:14:24,390 to carbohydrates or glycans. 253 00:14:24,390 --> 00:14:28,527 Are antibodies supravital or not, and why-- 254 00:14:28,527 --> 00:14:29,360 if they're not, why? 255 00:14:33,150 --> 00:14:35,970 Could I use that on a cell and follow a live cell 256 00:14:35,970 --> 00:14:36,820 with an antibody? 257 00:14:36,820 --> 00:14:37,320 Yeah? 258 00:14:37,320 --> 00:14:37,955 Carmen? 259 00:14:37,955 --> 00:14:39,747 AUDIENCE: It doesn't seem very likely to me 260 00:14:39,747 --> 00:14:42,348 that the antibodies will allow the proteins to do 261 00:14:42,348 --> 00:14:44,220 whatever it is that they do. 262 00:14:44,220 --> 00:14:45,303 PROFESSOR: Right. 263 00:14:45,303 --> 00:14:47,220 AUDIENCE: So I think that the cells would die. 264 00:14:47,220 --> 00:14:48,720 PROFESSOR: So it's a correct-- 265 00:14:48,720 --> 00:14:52,470 they are not supravital. 266 00:14:52,470 --> 00:14:54,420 What do you think would happen if I add 267 00:14:54,420 --> 00:14:57,420 an antibody to a living cell? 268 00:14:57,420 --> 00:15:01,080 Could it stain-- it could stain something on the cell surface, 269 00:15:01,080 --> 00:15:03,180 but it might, as you say, interfere 270 00:15:03,180 --> 00:15:04,890 with the function of the cell. 271 00:15:04,890 --> 00:15:08,130 Let's say I have an antibody to the epidermal growth factor 272 00:15:08,130 --> 00:15:09,090 receptor. 273 00:15:09,090 --> 00:15:11,040 That is going to alter the properties. 274 00:15:11,040 --> 00:15:13,230 So you're correct in that respect. 275 00:15:13,230 --> 00:15:17,780 What about targets inside cells? 276 00:15:17,780 --> 00:15:19,260 Are the antibodies going to get in? 277 00:15:19,260 --> 00:15:20,412 Yeah? 278 00:15:20,412 --> 00:15:21,394 AUDIENCE: [INAUDIBLE] 279 00:15:22,380 --> 00:15:23,850 PROFESSOR: Yeah. 280 00:15:23,850 --> 00:15:24,890 Yeah. 281 00:15:24,890 --> 00:15:26,710 Yeah, it's a big wall, frankly. 282 00:15:26,710 --> 00:15:30,130 It's an impenetrable barrier to get in. 283 00:15:30,130 --> 00:15:32,800 So you can really only stain in-- 284 00:15:32,800 --> 00:15:38,095 you can only observe in what are known as fixed cells. 285 00:15:42,010 --> 00:15:44,500 And when we call a cell a fixed cell, 286 00:15:44,500 --> 00:15:47,590 we actually mean we've broken it because what we've done 287 00:15:47,590 --> 00:15:50,810 is we've treated the cell population with methanol, 288 00:15:50,810 --> 00:15:53,390 which pokes holes all over the cellular membranes. 289 00:15:53,890 --> 00:15:54,640 They're fixed. 290 00:15:54,640 --> 00:15:56,410 They're static on a slide. 291 00:15:56,410 --> 00:15:58,480 They're not going to move around anymore, 292 00:15:58,480 --> 00:16:00,430 and you can stain with antibodies. 293 00:16:00,430 --> 00:16:02,830 You can tell what happened at the moment-- 294 00:16:02,830 --> 00:16:05,380 what was happening at the moment the cell 295 00:16:05,380 --> 00:16:07,670 passed away, if you will, what was going on, 296 00:16:07,670 --> 00:16:08,860 which proteins were there. 297 00:16:08,860 --> 00:16:11,530 But you can't keep observing moving forward 298 00:16:11,530 --> 00:16:14,770 because the cells then are no longer viable, all right? 299 00:16:14,770 --> 00:16:17,950 So neither of these approaches are super vital, 300 00:16:17,950 --> 00:16:20,030 but they're for different reasons. 301 00:16:20,030 --> 00:16:21,640 One is toxicity. 302 00:16:21,640 --> 00:16:24,880 The other one is also toxicity, as Carmen pointed out, 303 00:16:24,880 --> 00:16:27,940 but it's also a problem with membrane permeability. 304 00:16:27,940 --> 00:16:30,670 So what we're going to talk about in the rest 305 00:16:30,670 --> 00:16:33,820 of this lecture is a way to get around 306 00:16:33,820 --> 00:16:39,715 this impenetrable problem, and this-- 307 00:16:39,715 --> 00:16:49,020 and the discovery of the green fluorescent protein, 308 00:16:49,020 --> 00:16:53,530 and we're really mostly about GFP and its close siblings 309 00:16:53,530 --> 00:16:55,270 that might be different colors. 310 00:16:55,270 --> 00:16:57,460 What I want to walk you through is the discovery 311 00:16:57,460 --> 00:16:59,530 of the protein, and I really want 312 00:16:59,530 --> 00:17:04,240 to impress upon you how science happens in funny, small steps 313 00:17:04,240 --> 00:17:05,200 early on. 314 00:17:05,200 --> 00:17:09,220 You could never have predicted that a jellyfish 315 00:17:09,220 --> 00:17:11,170 off the coast of Seattle was going 316 00:17:11,170 --> 00:17:14,800 to revolutionize biology, biochemistry, life sciences, 317 00:17:14,800 --> 00:17:15,740 right? 318 00:17:15,740 --> 00:17:17,650 Who would have ever thought of that? 319 00:17:17,650 --> 00:17:22,210 And so that's why, you know, they colleagues, we 320 00:17:22,210 --> 00:17:25,210 and our colleagues, are so excited about fundamental 321 00:17:25,210 --> 00:17:28,160 science where you don't quite know where you're going, 322 00:17:28,160 --> 00:17:30,280 but you're working on something cool, 323 00:17:30,280 --> 00:17:33,820 and then the prepared mind goes, wow! 324 00:17:33,820 --> 00:17:35,020 That's interesting. 325 00:17:35,020 --> 00:17:37,720 I could use that for this problem. 326 00:17:37,720 --> 00:17:41,260 So Shimamura was a Japanese biochemist, 327 00:17:41,260 --> 00:17:46,360 who was fascinated by jellyfish and their bioluminescence 328 00:17:46,360 --> 00:17:49,810 and worked for years, slaved away for years and years. 329 00:17:49,810 --> 00:17:52,030 Apparently, he would go with his family 330 00:17:52,030 --> 00:17:54,670 to the small islands in Puget Sound 331 00:17:54,670 --> 00:17:57,670 and have his kids go and collect jellyfish all day 332 00:17:57,670 --> 00:18:01,540 long because he noticed certain things about the jellyfish that 333 00:18:01,540 --> 00:18:05,050 were rather intriguing with respect to their properties. 334 00:18:05,050 --> 00:18:07,150 And the key thing that was observed 335 00:18:07,150 --> 00:18:12,190 was that there is a bioluminescent protein 336 00:18:12,190 --> 00:18:14,830 in jellyfish known as aequorin. 337 00:18:14,830 --> 00:18:17,500 So that's the kind of luminescence we just 338 00:18:17,500 --> 00:18:21,460 described, but in the dark, there was also 339 00:18:21,460 --> 00:18:26,140 a fluorescence species, and it turns out 340 00:18:26,140 --> 00:18:29,140 that the light energy from a aequorin 341 00:18:29,140 --> 00:18:32,830 actually can excite the flourophore 342 00:18:32,830 --> 00:18:34,930 in the green fluorescent protein, 343 00:18:34,930 --> 00:18:40,030 and then you emit the light of the wavelength in the green. 344 00:18:40,030 --> 00:18:43,900 So it was actually a couple pair of proteins, aequorin 345 00:18:43,900 --> 00:18:45,910 and the second thing that was just 346 00:18:45,910 --> 00:18:49,270 the green fluorescent protein, and what was fascinating 347 00:18:49,270 --> 00:18:51,800 about this protein is more and more work was done 348 00:18:51,800 --> 00:18:54,610 is they didn't seem to need any additives. 349 00:18:54,610 --> 00:18:57,790 So for bioluminescence, you got to add ATP, 350 00:18:57,790 --> 00:19:02,800 and you've got to add, you know, the luciferin. 351 00:19:02,800 --> 00:19:05,650 You've got to add things to see the fluorescence. 352 00:19:05,650 --> 00:19:08,600 What was unique about the green fluorescent protein 353 00:19:08,600 --> 00:19:10,510 is it didn't need anything to add. 354 00:19:10,510 --> 00:19:12,922 You just had the protein, and it was fluorescent. 355 00:19:12,922 --> 00:19:14,380 So what I want to talk to you about 356 00:19:14,380 --> 00:19:17,430 is the molecular basis of this fluorescent, 357 00:19:17,430 --> 00:19:21,070 and we'll also talk, though, about the protein engineering 358 00:19:21,070 --> 00:19:24,220 that was systematically done to make this more and more 359 00:19:24,220 --> 00:19:26,590 of a useful reagent. 360 00:19:26,590 --> 00:19:29,360 So Shimamura was the first person. 361 00:19:29,360 --> 00:19:32,430 He had his kids collect so many jellyfish 362 00:19:32,430 --> 00:19:35,110 that they could extract the green fluorescent protein 363 00:19:35,110 --> 00:19:38,440 through traditional old-school biochemical methods 364 00:19:38,440 --> 00:19:41,530 and grow crystals, protein crystals. 365 00:19:41,530 --> 00:19:42,100 Guess what? 366 00:19:42,100 --> 00:19:46,000 They are bright green, and we're able to solve the structure. 367 00:19:46,000 --> 00:19:48,070 Once they had the structure, they sort of 368 00:19:48,070 --> 00:19:50,830 knew what was going on with the jellyfish, 369 00:19:50,830 --> 00:19:54,640 and they were also able to recognize a particular part 370 00:19:54,640 --> 00:19:57,880 of the sequence of the green fluorescent protein that 371 00:19:57,880 --> 00:20:00,760 ended up being the precursor to the fluorophore 372 00:20:00,760 --> 00:20:04,090 that we see nowadays and we understand. 373 00:20:04,090 --> 00:20:06,970 Originally, the protein was not monomeric. 374 00:20:06,970 --> 00:20:09,370 I'll talk to you a bit about that later. 375 00:20:09,370 --> 00:20:11,740 For technology, it's much more handy to have 376 00:20:11,740 --> 00:20:15,340 this protein as a monomore, not as a dimer or a tetramer. 377 00:20:15,340 --> 00:20:17,720 That makes experiments complicated, 378 00:20:17,720 --> 00:20:19,420 but I'll show you a very easy trick 379 00:20:19,420 --> 00:20:21,410 that was done to fix that. 380 00:20:21,410 --> 00:20:25,290 And so the other people who shared Nobel Prize 381 00:20:25,290 --> 00:20:27,790 with Shimamura, who, by the way, passed away just a couple 382 00:20:27,790 --> 00:20:30,730 of weeks ago, in fact, but a an amazing old age. 383 00:20:30,730 --> 00:20:33,190 It must have been all that digging for jellyfish 384 00:20:33,190 --> 00:20:35,650 that helped him live that long. 385 00:20:35,650 --> 00:20:38,620 There was also Martin Chalfie at Columbia, 386 00:20:38,620 --> 00:20:42,340 who demonstrated that the gene for the green fluorescent 387 00:20:42,340 --> 00:20:46,040 protein could be put in all kinds of other animals, 388 00:20:46,040 --> 00:20:49,930 organisms, C elegans bacteria and they would fluoresce. 389 00:20:49,930 --> 00:20:54,220 And a really major player in this entire story 390 00:20:54,220 --> 00:20:59,980 was Roger Tsien, who died very young of a stroke, who 391 00:20:59,980 --> 00:21:04,540 was the chemist-biochemist who put the pieces together said, 392 00:21:04,540 --> 00:21:08,640 if this is GFP, we can use it for so many different things. 393 00:21:08,640 --> 00:21:11,680 So he really advanced the technologies 394 00:21:11,680 --> 00:21:13,840 of the applications. 395 00:21:13,840 --> 00:21:17,920 So protein expression could be-- they realized quite early on 396 00:21:17,920 --> 00:21:20,590 could just be programmed into a protein 397 00:21:20,590 --> 00:21:24,190 by just having the DNA for the green fluorescent protein stuck 398 00:21:24,190 --> 00:21:27,040 on to a favorite protein of interest. 399 00:21:27,040 --> 00:21:31,510 Then you would have that DNA be transcribed, translated 400 00:21:31,510 --> 00:21:35,410 in the cell, and then your favorite protein in the cell 401 00:21:35,410 --> 00:21:37,750 would fluoresce green because it was 402 00:21:37,750 --> 00:21:40,590 attached as a construct with the other proteins. 403 00:21:40,590 --> 00:21:44,590 So all of those things became enabled quite quickly. 404 00:21:44,590 --> 00:21:48,800 This could be done in all organisms, eukaryotes, 405 00:21:48,800 --> 00:21:49,390 prokaryotes. 406 00:21:49,390 --> 00:21:54,400 It's pretty non-toxic, so expressing a bunch of GFP 407 00:21:54,400 --> 00:21:55,840 in a cell doesn't kill it. 408 00:21:55,840 --> 00:21:57,110 That's kind of handy. 409 00:21:57,110 --> 00:22:00,880 So it really is a super vital system, 410 00:22:00,880 --> 00:22:03,070 and it's visible in all kinds of tissues. 411 00:22:03,070 --> 00:22:05,280 So what I'm going to show you here 412 00:22:05,280 --> 00:22:09,130 are some of the seminal results, first of all, the structure 413 00:22:09,130 --> 00:22:12,070 we'll talk about and then Chalfie 414 00:22:12,070 --> 00:22:16,450 was able to put the DNA into proteins and bacteria, 415 00:22:16,450 --> 00:22:21,250 and, also, proteins that were labeled in C elegans. 416 00:22:21,250 --> 00:22:23,830 And the story of Chalfie is kind of funny. 417 00:22:23,830 --> 00:22:26,770 He's one of those guys who sort of didn't necessarily 418 00:22:26,770 --> 00:22:28,330 have all the equipment he needed, 419 00:22:28,330 --> 00:22:30,010 but he knew this was really exciting, 420 00:22:30,010 --> 00:22:32,260 and he needed a fluorescence microscope. 421 00:22:32,260 --> 00:22:34,180 So he would call up all the companies who 422 00:22:34,180 --> 00:22:36,430 sold microscopes and, say, I'm really 423 00:22:36,430 --> 00:22:39,190 thinking of buying this fluorescent microscope. 424 00:22:39,190 --> 00:22:41,560 This particular one would be-- 425 00:22:41,560 --> 00:22:43,240 these are a couple of hundred grand, 426 00:22:43,240 --> 00:22:45,020 you know, these are not cheap things. 427 00:22:45,020 --> 00:22:48,970 And he'd talk the company into putting a microscope in his lab 428 00:22:48,970 --> 00:22:50,560 for a month as a demo. 429 00:22:50,560 --> 00:22:53,230 It's, like, you know, saying to the car 430 00:22:53,230 --> 00:22:56,230 dealer you want to buy a car and then driving a Ferrari around 431 00:22:56,230 --> 00:22:58,090 for a month and then saying, it's not really 432 00:22:58,090 --> 00:22:59,300 going to work for me. 433 00:22:59,300 --> 00:23:01,900 So he collected all his early data 434 00:23:01,900 --> 00:23:05,580 on microscopes that were on loan from Olympus and Leica 435 00:23:05,580 --> 00:23:07,040 and various other places. 436 00:23:07,040 --> 00:23:09,270 So that's a funny, funny twist. 437 00:23:09,270 --> 00:23:11,440 OK, so the fluorophore-- 438 00:23:11,440 --> 00:23:17,380 all right, so they could go from DNA to protein sequence. 439 00:23:17,380 --> 00:23:19,360 Then they could look at the structure 440 00:23:19,360 --> 00:23:23,740 and see that the place in the protein sequence that went 441 00:23:23,740 --> 00:23:26,530 serine, tyrosine, glycine. 442 00:23:26,530 --> 00:23:29,900 So here we go-- serine, tyrosine. 443 00:23:29,900 --> 00:23:31,020 You recognize that one. 444 00:23:31,020 --> 00:23:33,410 It's the one with one of the aromatic rings, 445 00:23:33,410 --> 00:23:35,750 glycine is the one with no substituent. 446 00:23:35,750 --> 00:23:40,280 It was common in a number of organisms, 447 00:23:40,280 --> 00:23:43,880 mostly aquatic organisms that fluoresce and seem 448 00:23:43,880 --> 00:23:47,375 to be the origin of the flourophore in GFP, 449 00:23:47,375 --> 00:23:50,000 and so the chemists got to work, and you know what chemists do, 450 00:23:50,000 --> 00:23:53,750 they start drawing arrows and joining bonds and figuring out 451 00:23:53,750 --> 00:23:56,990 how can we go from something that's basically dead, 452 00:23:56,990 --> 00:23:58,760 and it's not got any fluorescence at all 453 00:23:58,760 --> 00:24:00,710 for something that's fluorescent. 454 00:24:00,710 --> 00:24:05,600 And so from the structure and from working out the mechanism, 455 00:24:05,600 --> 00:24:09,890 they basically found that this little piece, this tripeptide 456 00:24:09,890 --> 00:24:13,970 within the entire structure of the DNA primary sequence, 457 00:24:13,970 --> 00:24:17,840 was able to cyclize through that nitrogen attacking 458 00:24:17,840 --> 00:24:20,690 that carbon and then an elimination, 459 00:24:20,690 --> 00:24:25,460 and then there was an oxygen-dependent oxidation 460 00:24:25,460 --> 00:24:27,590 to give you this structure. 461 00:24:27,590 --> 00:24:31,130 So if you look at this, you can still see the serine. 462 00:24:31,130 --> 00:24:32,870 You kind of know this was tyrosine, 463 00:24:32,870 --> 00:24:34,850 but it doesn't look like it anymore, 464 00:24:34,850 --> 00:24:37,990 and it turns out the glycine was incredibly important. 465 00:24:37,990 --> 00:24:41,480 It doesn't look like it's involved in the chromophore, 466 00:24:41,480 --> 00:24:43,820 but when you have a glycine in a sequence, 467 00:24:43,820 --> 00:24:45,680 it allows you to do some funny twists 468 00:24:45,680 --> 00:24:48,320 and turns because it has no substituents. 469 00:24:48,320 --> 00:24:50,960 So it allowed that loop to form. 470 00:24:50,960 --> 00:24:55,130 Once that loop is formed, chemistry can occur. 471 00:24:55,130 --> 00:24:57,050 If the glycine wasn't there, things 472 00:24:57,050 --> 00:24:59,840 might be not in an ideal situation 473 00:24:59,840 --> 00:25:01,460 to form the fluorophore. 474 00:25:01,460 --> 00:25:04,680 The oxidation to put in that double bond 475 00:25:04,680 --> 00:25:06,350 is oxygen dependent. 476 00:25:06,350 --> 00:25:08,570 So in some cases, the fluorophore 477 00:25:08,570 --> 00:25:10,980 would mature a bit more slowly. 478 00:25:10,980 --> 00:25:13,280 So you would go from the free state that's 479 00:25:13,280 --> 00:25:16,160 not fluorescent to the fluorescent state 480 00:25:16,160 --> 00:25:19,160 quite slowly if you withheld oxygen. 481 00:25:19,160 --> 00:25:23,480 So a lot of engineering was done to improve the maturation time, 482 00:25:23,480 --> 00:25:25,260 to improve the photo physics. 483 00:25:25,260 --> 00:25:27,740 There's really cool photophysical experiments that 484 00:25:27,740 --> 00:25:30,650 were done, but that's the basis of it, 485 00:25:30,650 --> 00:25:33,028 and the emission of this flouorophore, 486 00:25:33,028 --> 00:25:35,570 you can almost recognize that it's a fluorophore because it's 487 00:25:35,570 --> 00:25:38,090 got a lot of what's called conjugation 488 00:25:38,090 --> 00:25:39,920 in organic chemistry-- 489 00:25:39,920 --> 00:25:41,870 one double bond, another double bond 490 00:25:41,870 --> 00:25:44,720 stuck onto this ring with multiple double bonds. 491 00:25:44,720 --> 00:25:47,840 And it emits at the same-- a similar wavelength 492 00:25:47,840 --> 00:25:52,190 to the fluorophore fluoroscein, so people were happy 493 00:25:52,190 --> 00:25:57,200 because all the filters on their microscopes 494 00:25:57,200 --> 00:25:58,330 were the right ones. 495 00:25:58,330 --> 00:26:02,060 If you just used the fluoroscein filters, you would get a GFPC. 496 00:26:02,060 --> 00:26:04,800 So that really was made in heaven for those guys. 497 00:26:04,800 --> 00:26:06,210 OK, so let's take a look at this. 498 00:26:06,210 --> 00:26:09,020 Here's the wire diagram of the original crystallized 499 00:26:09,020 --> 00:26:10,010 structure. 500 00:26:10,010 --> 00:26:12,050 I've planted a ribbon on there. 501 00:26:12,050 --> 00:26:13,520 I get rid of all the side chains, 502 00:26:13,520 --> 00:26:16,130 so you see this beautiful dimer structure. 503 00:26:16,130 --> 00:26:18,320 Let's throw away one of the dimers, 504 00:26:18,320 --> 00:26:19,430 and you can start to look. 505 00:26:19,430 --> 00:26:22,490 There's a little something sneaky in there, 506 00:26:22,490 --> 00:26:26,690 and as you get closer, you can start to see the structure. 507 00:26:26,690 --> 00:26:28,970 There's this sort of thread going through it. 508 00:26:28,970 --> 00:26:31,250 That's just a trace of the backbone, 509 00:26:31,250 --> 00:26:33,880 but there is the structure of the fluorophore. 510 00:26:33,880 --> 00:26:36,290 Here's the fluorophore with the space filling, 511 00:26:36,290 --> 00:26:39,320 and what I think is kind of cool is if you kind of twist 512 00:26:39,320 --> 00:26:42,110 the GFP around, it's kind of like a barrel, 513 00:26:42,110 --> 00:26:43,790 and it looks like a bird in a cage 514 00:26:43,790 --> 00:26:45,830 because it's caged in there. 515 00:26:45,830 --> 00:26:49,280 And to be honest, it's the structure. 516 00:26:49,280 --> 00:26:51,470 It's not just the structure of the flourophore. 517 00:26:51,470 --> 00:26:54,790 It's the fact that the fluorophore is inside 518 00:26:54,790 --> 00:26:56,810 a kind of hydrophobic environment 519 00:26:56,810 --> 00:26:58,560 that makes it fluoresce. 520 00:26:58,560 --> 00:27:02,240 I could go into the lab and make that flourophore 521 00:27:02,240 --> 00:27:03,830 and have it in a beaker of methanol, 522 00:27:03,830 --> 00:27:05,450 and you wouldn't see anything. 523 00:27:05,450 --> 00:27:09,110 It's only in that environment that's created in the GFP 524 00:27:09,110 --> 00:27:11,510 molecule, so it's fascinating. 525 00:27:11,510 --> 00:27:16,610 The structure of GFP creates the shape to cyclize, 526 00:27:16,610 --> 00:27:20,300 and it creates the environment for good fluorescence. 527 00:27:20,300 --> 00:27:24,260 So he has a mass of animals that have been labeled with GFP. 528 00:27:24,260 --> 00:27:27,440 As you've seen, our mouse has been labeled multiple times. 529 00:27:27,440 --> 00:27:30,080 This was the first GFP rabbit. 530 00:27:30,080 --> 00:27:31,400 He was known as Alba. 531 00:27:31,400 --> 00:27:34,730 He even had his own name, but there's 532 00:27:34,730 --> 00:27:41,210 seaweeds and zebrafish and mice from a letter, C elegans, 533 00:27:41,210 --> 00:27:43,880 the famous, the fly. 534 00:27:43,880 --> 00:27:44,960 What else? 535 00:27:44,960 --> 00:27:49,530 I don't even know what these things are, but Purkinje cells 536 00:27:49,530 --> 00:27:50,030 and so on. 537 00:27:50,030 --> 00:27:54,980 Anyway, so this shows you the universality of the tool. 538 00:27:54,980 --> 00:27:58,607 A brief mention about the dimer problem, people 539 00:27:58,607 --> 00:28:00,190 looked at the structure of the diamond 540 00:28:00,190 --> 00:28:02,240 from the crystal structure and found 541 00:28:02,240 --> 00:28:06,320 that there was a sticky face between two monomers of GFP 542 00:28:06,320 --> 00:28:09,500 that encouraged this quaternary structure. 543 00:28:09,500 --> 00:28:12,710 And so they simply changed two alanines that 544 00:28:12,710 --> 00:28:15,110 were sitting right at the dimer interface, one 545 00:28:15,110 --> 00:28:17,300 from one monomer and one from the other, 546 00:28:17,300 --> 00:28:18,560 created a sticky patch. 547 00:28:18,560 --> 00:28:21,590 Sounds a bit like hemoglobin, if you remember, 548 00:28:21,590 --> 00:28:23,330 the sickle cell hemoglobin. 549 00:28:23,330 --> 00:28:26,060 They changed them to lycines and [INAUDIBLE] 550 00:28:26,060 --> 00:28:30,130 solved you had just monomeric protein 551 00:28:30,130 --> 00:28:32,830 because when you do a lot of experiments with GFP, 552 00:28:32,830 --> 00:28:36,610 you don't want GFP randomly dimerizing because it's 553 00:28:36,610 --> 00:28:40,270 going to change the biology of the protein it's attached to. 554 00:28:40,270 --> 00:28:44,920 So this very, very easy structure-driven engineering 555 00:28:44,920 --> 00:28:46,420 was perfect. 556 00:28:46,420 --> 00:28:49,300 OK, so let's take a look at some of the early things that 557 00:28:49,300 --> 00:28:50,050 could be done now. 558 00:28:50,050 --> 00:28:52,870 We take it all for granted, but GFP 559 00:28:52,870 --> 00:28:57,290 can be used as a reporter gene to do a number of things 560 00:28:57,290 --> 00:29:00,200 in cellular systems. 561 00:29:00,200 --> 00:29:05,140 So, for example, if you want to study a regulatory sequence 562 00:29:05,140 --> 00:29:07,510 to know whether this promoter works well, 563 00:29:07,510 --> 00:29:11,170 you can put it at the front end of the GFP gene 564 00:29:11,170 --> 00:29:14,890 and then see if the promoter works under certain conditions, 565 00:29:14,890 --> 00:29:16,690 then the GFP will be produced. 566 00:29:16,690 --> 00:29:19,170 The cells will grow green. 567 00:29:19,170 --> 00:29:24,250 And this is a nice variant to a luciferase-based system 568 00:29:24,250 --> 00:29:27,010 or a LacZ based system where you're actually 569 00:29:27,010 --> 00:29:29,560 having to treat and fix things in order 570 00:29:29,560 --> 00:29:32,590 to do the LacZ experiments. 571 00:29:32,590 --> 00:29:36,880 So here's a really nice example with C elegans. 572 00:29:36,880 --> 00:29:38,620 What you could do pre-GF-- 573 00:29:38,620 --> 00:29:40,960 we really have-- it's like ADBC-- 574 00:29:40,960 --> 00:29:47,620 you know, anti whatever before year 0 and after year 0. 575 00:29:47,620 --> 00:29:51,960 The biological world is pre GFP and post GFP, frankly, 576 00:29:51,960 --> 00:29:54,830 the impact that it's had on the system. 577 00:29:54,830 --> 00:29:56,440 So here is a C elegans. 578 00:29:56,440 --> 00:29:59,800 As you know, all the neurons have names, and a lot of C 579 00:29:59,800 --> 00:30:02,890 elegans biologists are very familiar with each, 580 00:30:02,890 --> 00:30:06,010 you know, single cell within the C elegans, in particular, 581 00:30:06,010 --> 00:30:07,060 the neurons. 582 00:30:07,060 --> 00:30:12,800 So this is a listing of the touch receptors. 583 00:30:12,800 --> 00:30:15,580 You could have an antibody to those, 584 00:30:15,580 --> 00:30:18,400 and so you could do some antibody staining, 585 00:30:18,400 --> 00:30:21,910 but antibody staining, remember, we got to fix the worms. 586 00:30:21,910 --> 00:30:22,830 They don't like that. 587 00:30:22,830 --> 00:30:26,200 You literally fix them down and do the staining 588 00:30:26,200 --> 00:30:29,380 with the antibody to get inside the C elegans 589 00:30:29,380 --> 00:30:32,800 to see the antibody light up the proteins. 590 00:30:32,800 --> 00:30:37,870 You could do beta-gal activity, same thing, but in both cases, 591 00:30:37,870 --> 00:30:39,520 these are dead C elegans. 592 00:30:39,520 --> 00:30:40,312 They're fixed. 593 00:30:40,312 --> 00:30:41,020 They're in place. 594 00:30:41,020 --> 00:30:43,540 You can't observe them going forward. 595 00:30:43,540 --> 00:30:45,700 But look instead, that the images you 596 00:30:45,700 --> 00:30:49,270 would get with GFP, where you could totally pick out 597 00:30:49,270 --> 00:30:51,050 all the touch receptors. 598 00:30:51,050 --> 00:30:54,190 And this is a situation where an important protein 599 00:30:54,190 --> 00:30:59,890 in those cells MEC-17 is co expressed with GFP, 600 00:30:59,890 --> 00:31:01,960 and you can see this beautiful image, 601 00:31:01,960 --> 00:31:03,280 and the worms are still alive. 602 00:31:03,280 --> 00:31:06,770 You could totally watch what was going on with them, 603 00:31:06,770 --> 00:31:08,770 and he just wants you to know that he's 604 00:31:08,770 --> 00:31:10,140 alive and well and kicking. 605 00:31:10,140 --> 00:31:15,650 OK, now, progress has to happen. 606 00:31:15,650 --> 00:31:19,420 So one green protein does not a history make. 607 00:31:19,420 --> 00:31:22,720 So, immediately, with the identity 608 00:31:22,720 --> 00:31:24,860 of the structure of GFP, I am going 609 00:31:24,860 --> 00:31:26,530 to go back really quickly to look 610 00:31:26,530 --> 00:31:29,320 at that picture of the structure for you. 611 00:31:29,320 --> 00:31:31,030 Sorry about this back and forward. 612 00:31:31,030 --> 00:31:36,580 OK, the chemists realize that if they change this tyrosine 613 00:31:36,580 --> 00:31:38,380 to some of the other amino acids that 614 00:31:38,380 --> 00:31:42,220 looked a little bit like it, like phenylalanine, tryptophan, 615 00:31:42,220 --> 00:31:46,750 and histidine, they might alter the photophysics 616 00:31:46,750 --> 00:31:49,600 of this structure because if you put tryptophan there, 617 00:31:49,600 --> 00:31:51,670 there's even more double bonds. 618 00:31:51,670 --> 00:31:55,540 If you put phenylalanine there, that OH is gone. 619 00:31:55,540 --> 00:31:58,918 So there's really opportunities, and that's the chemists saying, 620 00:31:58,918 --> 00:32:00,460 I can look at this protein structure, 621 00:32:00,460 --> 00:32:02,620 and I know the one thing I can change 622 00:32:02,620 --> 00:32:06,610 is to change that amino acid back at the DNA level. 623 00:32:06,610 --> 00:32:10,690 So I'm going to change the codon for tyrosine to the codons 624 00:32:10,690 --> 00:32:14,200 for tryptophan, phenylalanine, and histidine 625 00:32:14,200 --> 00:32:17,380 and then see what my fluorescent protein looks like, OK? 626 00:32:17,380 --> 00:32:18,580 Does that make sense? 627 00:32:18,580 --> 00:32:20,560 So it was the one thing that's obvious. 628 00:32:20,560 --> 00:32:24,090 I can fix this. 629 00:32:24,090 --> 00:32:26,970 So what they did was they made the series of GFPs, 630 00:32:26,970 --> 00:32:29,130 and they transfected cells, and they 631 00:32:29,130 --> 00:32:32,910 were able to see that when you replaced 632 00:32:32,910 --> 00:32:40,650 various residues, for example, tyrosine to tryptophan, 633 00:32:40,650 --> 00:32:42,210 tyrosine to histidine. 634 00:32:42,210 --> 00:32:45,600 There's a little bit of variation here, 635 00:32:45,600 --> 00:32:47,790 and don't worry about this for a minute. 636 00:32:47,790 --> 00:32:51,570 They were able to create bacteria 637 00:32:51,570 --> 00:32:55,440 that had been transfected with the mutated GFPs 638 00:32:55,440 --> 00:32:58,650 in order to give them different colors of bacteria. 639 00:32:58,650 --> 00:33:01,590 So my question to you is, if you look at these, this 640 00:33:01,590 --> 00:33:03,720 would have been the original GFP, 641 00:33:03,720 --> 00:33:05,340 except made a little bit brighter 642 00:33:05,340 --> 00:33:08,880 by a small substitution, which of these would emit 643 00:33:08,880 --> 00:33:10,530 at the shortest wavelengths. 644 00:33:10,530 --> 00:33:12,480 So just look at the colors, which 645 00:33:12,480 --> 00:33:14,940 is the shortest wavelength, which 646 00:33:14,940 --> 00:33:17,610 is the bacterium that expresses the protein 647 00:33:17,610 --> 00:33:21,930 that emits at the shortest wavelength. 648 00:33:21,930 --> 00:33:24,000 You'd always be provided a picture 649 00:33:24,000 --> 00:33:27,520 of the electromagnetic spectrum. 650 00:33:27,520 --> 00:33:29,850 Yeah, out there. 651 00:33:29,850 --> 00:33:30,650 AUDIENCE: The GFP. 652 00:33:30,650 --> 00:33:32,450 PROFESSOR: Yeah, the blue one, exactly. 653 00:33:32,450 --> 00:33:34,970 So you can look at the spectrum, and you say, OK, I've 654 00:33:34,970 --> 00:33:37,050 got blue down here. 655 00:33:37,050 --> 00:33:38,150 There's that cyan. 656 00:33:38,150 --> 00:33:40,310 It's kind of a blue green, and then I 657 00:33:40,310 --> 00:33:42,260 move towards the more yellow. 658 00:33:42,260 --> 00:33:46,410 So you can pick it out and say, it's the lowest energy. 659 00:33:46,410 --> 00:33:50,180 It's the shortest wavelength emission, the highest energy 660 00:33:50,180 --> 00:33:51,410 emission. 661 00:33:51,410 --> 00:33:54,320 OK, all right, and these were the variance 662 00:33:54,320 --> 00:33:56,930 that I just described while I went back to the picture. 663 00:33:56,930 --> 00:34:00,050 So you could make a blue one with histidine. 664 00:34:00,050 --> 00:34:03,440 You could make a cyan one with tryptophan. 665 00:34:03,440 --> 00:34:07,280 You could preserve the green one with tyrosine, 666 00:34:07,280 --> 00:34:10,294 but a change in the serine to a threonine just 667 00:34:10,294 --> 00:34:12,980 to improve the photophysics, a long story. 668 00:34:12,980 --> 00:34:15,300 I won't-- I'm happy to chat about it. 669 00:34:15,300 --> 00:34:17,239 And, in fact, there was one clever one 670 00:34:17,239 --> 00:34:20,290 where you couldn't really make the protein yellow, 671 00:34:20,290 --> 00:34:23,690 but if you had the tyrosine nearby sandwiched 672 00:34:23,690 --> 00:34:26,420 with the tyrosine and the GFP chromophore, 673 00:34:26,420 --> 00:34:29,250 you could actually go as far as the yellow flouorphore. 674 00:34:29,250 --> 00:34:32,750 And that, in principle, should make everybody really thrilled. 675 00:34:32,750 --> 00:34:36,052 It's always this situation with protein engineering 676 00:34:36,052 --> 00:34:38,260 where you make something, and it's a huge improvement 677 00:34:38,260 --> 00:34:41,150 and then everyone says, well, what else, you know? 678 00:34:41,150 --> 00:34:42,510 What's next? 679 00:34:42,510 --> 00:34:43,760 We need red dyes. 680 00:34:43,760 --> 00:34:46,790 We need all kinds of different dyes. 681 00:34:46,790 --> 00:34:50,780 So teams went back to the oceans and actually 682 00:34:50,780 --> 00:34:54,679 collected organisms based on the color they fluoresced 683 00:34:54,679 --> 00:34:58,700 at, nice way to sample things. 684 00:34:58,700 --> 00:35:02,690 And, actually, we were able to ultimately discover 685 00:35:02,690 --> 00:35:06,710 a red fluorescent protein from the Discosoma 686 00:35:06,710 --> 00:35:09,830 coral, the original protein, DS red, 687 00:35:09,830 --> 00:35:11,580 and if you look at this kind of carefully, 688 00:35:11,580 --> 00:35:13,550 it looks a lot like GFP. 689 00:35:13,550 --> 00:35:15,867 There's the tyrosine, that funny ring. 690 00:35:15,867 --> 00:35:17,450 There's a double bond there, but there 691 00:35:17,450 --> 00:35:21,500 is an additional double bond down further 692 00:35:21,500 --> 00:35:26,270 into the sequence that extends what we call the chromophore. 693 00:35:26,270 --> 00:35:27,770 And when you extend the chromophore, 694 00:35:27,770 --> 00:35:30,920 you're more likely to move to longer wavelengths, 695 00:35:30,920 --> 00:35:34,220 and that's how they got the red, and then a bunch of engineering 696 00:35:34,220 --> 00:35:37,820 later, they got what's called the fruits, which emit 697 00:35:37,820 --> 00:35:40,130 at all different wavelengths. 698 00:35:40,130 --> 00:35:42,500 Much of this was not done rationally 699 00:35:42,500 --> 00:35:43,670 because they went to nature. 700 00:35:43,670 --> 00:35:45,470 They found out what nature did. 701 00:35:45,470 --> 00:35:47,990 They got here, and then they did a process 702 00:35:47,990 --> 00:35:50,270 known as gene shuffling, where they mixed 703 00:35:50,270 --> 00:35:53,090 and matched portions of genes to get them 704 00:35:53,090 --> 00:35:55,160 the entire color spectrum. 705 00:35:55,160 --> 00:35:57,860 They're not all fabulous fluorophores. 706 00:35:57,860 --> 00:35:59,060 They have some problems. 707 00:35:59,060 --> 00:36:01,940 They bleach easily and so on, but, nevertheless, this 708 00:36:01,940 --> 00:36:06,150 is a truly amazing sort of outcome. 709 00:36:06,150 --> 00:36:09,770 OK, so and they enable you to do art work 710 00:36:09,770 --> 00:36:12,830 with fluorescent proteins with bacteria, 711 00:36:12,830 --> 00:36:16,280 so you just paint your picture and wait for the bacteria 712 00:36:16,280 --> 00:36:20,270 to grow, and you would have all those colors. 713 00:36:20,270 --> 00:36:23,150 So fluorescent proteins originally 714 00:36:23,150 --> 00:36:26,720 were very limited, originally just green, then blue-green 715 00:36:26,720 --> 00:36:29,900 and greenish yellow, but, now, with the palette 716 00:36:29,900 --> 00:36:32,060 that's available, you can color all kinds 717 00:36:32,060 --> 00:36:35,530 of organelles in different types of colors, all right? 718 00:36:35,530 --> 00:36:38,690 OK, so now let's take a couple of things. 719 00:36:38,690 --> 00:36:41,420 When we opened up this class, we showed you 720 00:36:41,420 --> 00:36:43,310 a bunch of these pictures because we wanted 721 00:36:43,310 --> 00:36:47,660 you to stay in 7016 with us to see the cool things you'd see, 722 00:36:47,660 --> 00:36:50,790 and they were pretty much what we would call eye candy. 723 00:36:50,790 --> 00:36:53,690 They look cool, but how did we make them? 724 00:36:53,690 --> 00:36:58,010 And, now, with the knowledge of GFP and RFP and so on, 725 00:36:58,010 --> 00:37:01,592 you can say, OK, I understand what this experiment is, 726 00:37:01,592 --> 00:37:03,050 and you can certainly see that this 727 00:37:03,050 --> 00:37:05,250 is a super vital technique. 728 00:37:05,250 --> 00:37:07,880 So what I want to ask is, what are we looking at? 729 00:37:07,880 --> 00:37:11,690 What is labeled to give us this beautiful picture? 730 00:37:11,690 --> 00:37:15,290 So you can sort of go down, and there's a bunch of options, 731 00:37:15,290 --> 00:37:17,570 but, obviously, anything where you 732 00:37:17,570 --> 00:37:21,200 think you're labeling DNA with a fluorescent protein 733 00:37:21,200 --> 00:37:22,080 is incorrect. 734 00:37:22,080 --> 00:37:24,560 We're labeling at the protein level. 735 00:37:24,560 --> 00:37:28,160 We're making the DNA to express the fluorescent protein, 736 00:37:28,160 --> 00:37:32,700 but we're actually labeling a protein as opposed to DNA. 737 00:37:32,700 --> 00:37:37,340 So A and B are out for sure, but as we go through them, 738 00:37:37,340 --> 00:37:41,030 you can start to see the chromatin is red 739 00:37:41,030 --> 00:37:46,530 and the tubulin is green, so the outcome there would be, 740 00:37:46,530 --> 00:37:51,900 whoops, what we're observing-- 741 00:37:51,900 --> 00:37:55,890 I thought what we're observing is that the histones, which 742 00:37:55,890 --> 00:38:02,070 are the proteins associated with chromatin, 743 00:38:02,070 --> 00:38:03,675 are labeled with a red protein. 744 00:38:03,675 --> 00:38:04,800 And you can see that there. 745 00:38:04,800 --> 00:38:07,260 Those are the chromatids, and the tubulin, 746 00:38:07,260 --> 00:38:11,070 these green fibrils, is associated with a GFP. 747 00:38:11,070 --> 00:38:15,450 So C would be the correct answer there. 748 00:38:15,450 --> 00:38:18,390 And there's another one of these cell division pictures. 749 00:38:18,390 --> 00:38:21,150 Once again, the same sort of idea, but what I think 750 00:38:21,150 --> 00:38:24,775 is really cool here, as you can literally see, 751 00:38:24,775 --> 00:38:27,150 I love this picture because there's this poor chromosome. 752 00:38:27,150 --> 00:38:29,310 It doesn't seem to be able to get with the team. 753 00:38:29,310 --> 00:38:31,680 He's hanging down there, and, eventually, just 754 00:38:31,680 --> 00:38:35,670 before cell division, it seems like things work out OK 755 00:38:35,670 --> 00:38:39,120 and cell division happens. 756 00:38:39,120 --> 00:38:42,180 Now when you think of this, the capacity 757 00:38:42,180 --> 00:38:46,530 to watch this stuff in action is really pretty amazing. 758 00:38:46,530 --> 00:38:49,180 This was another one. 759 00:38:49,180 --> 00:38:53,340 This a set of cell lines where proteins in cell cycle 760 00:38:53,340 --> 00:38:54,450 are labeled. 761 00:38:54,450 --> 00:38:57,630 You had a question on exam three about proteins 762 00:38:57,630 --> 00:38:59,460 building up and going-- 763 00:38:59,460 --> 00:39:03,010 and going away during parts of the cell cycle. 764 00:39:03,010 --> 00:39:07,470 So, oftentimes, the degradation of a protein 765 00:39:07,470 --> 00:39:10,830 occurs when a protein gets labeled with ubiquitin 766 00:39:10,830 --> 00:39:13,310 by a ubiquitin ligase, and then it 767 00:39:13,310 --> 00:39:15,640 gets destined for degradation. 768 00:39:15,640 --> 00:39:18,390 So the proteins that you're seeing grow up 769 00:39:18,390 --> 00:39:20,880 in phases of the cell cycle are going away 770 00:39:20,880 --> 00:39:23,790 because of the ubiquitin proteasome system. 771 00:39:23,790 --> 00:39:27,720 So in these cells, particular proteins have been labeled, 772 00:39:27,720 --> 00:39:31,320 and here you can see the phases-- 773 00:39:31,320 --> 00:39:34,440 the components of the cell cycle as proteins 774 00:39:34,440 --> 00:39:37,170 come and go through cell division, 775 00:39:37,170 --> 00:39:40,320 and you can literally observe where in the cell cycle 776 00:39:40,320 --> 00:39:42,390 each individual protein is. 777 00:39:42,390 --> 00:39:44,790 Why is this so fascinating, I think, 778 00:39:44,790 --> 00:39:47,880 in therapeutic development is that you could literally 779 00:39:47,880 --> 00:39:52,200 have drugs that might impact aspects of the cell cycle, 780 00:39:52,200 --> 00:39:56,310 and you could watch them in real time impact the imaging 781 00:39:56,310 --> 00:39:59,130 that you see on this screen with the red and the green 782 00:39:59,130 --> 00:40:02,550 because if you arrest at a certain stage in cell cycle, 783 00:40:02,550 --> 00:40:05,280 the cell will get stuck at a particular color, 784 00:40:05,280 --> 00:40:08,160 and you would even know which phases, which parts of the cell 785 00:40:08,160 --> 00:40:10,450 cycle, are being impacted. 786 00:40:10,450 --> 00:40:13,050 So I think that's a really sort of captivating way 787 00:40:13,050 --> 00:40:15,840 to think of what you can do with these proteins 788 00:40:15,840 --> 00:40:19,470 because you can see things in real time, 789 00:40:19,470 --> 00:40:22,550 dynamics of cellular systems. 790 00:40:22,550 --> 00:40:26,670 And I think, oh, and I'm going to finish a few minutes early, 791 00:40:26,670 --> 00:40:30,390 but I looked desperately for a fluorescent turkey, 792 00:40:30,390 --> 00:40:31,770 and I couldn't find one. 793 00:40:31,770 --> 00:40:36,810 So I went with this light string turkey and fluorescent colors. 794 00:40:36,810 --> 00:40:38,010 It seemed to fit the bill. 795 00:40:38,010 --> 00:40:41,940 All right, so please help us finish up the bagels. 796 00:40:41,940 --> 00:40:46,200 Make sure you guys over there get them and have a good break, 797 00:40:46,200 --> 00:40:49,770 and I'm passing you back to Professor Martin 798 00:40:49,770 --> 00:40:55,430 for Monday, which will be a continuation of imaging, OK.