1 00:00:06,318 --> 00:00:07,290 ERIC LANDER: That worked. 2 00:00:07,290 --> 00:00:08,800 Let's do it again. 3 00:00:08,800 --> 00:00:13,970 Now, number 2. 4 00:00:13,970 --> 00:00:19,790 I would like to clone not the Arg1 gene, but let's say the 5 00:00:19,790 --> 00:00:23,020 beta globin gene from human. 6 00:00:23,020 --> 00:00:26,290 So let's take human beta globin. 7 00:00:31,710 --> 00:00:33,070 I want to clone it. 8 00:00:33,070 --> 00:00:37,810 So human beta globin, hemoglobin, it's a tetromer. 9 00:00:37,810 --> 00:00:39,530 It has four parts. 10 00:00:39,530 --> 00:00:42,070 It's got an alpha, an alpha, a beta, and a beta. 11 00:00:42,070 --> 00:00:44,930 Four proteins come together in a protein tetromer. 12 00:00:44,930 --> 00:00:46,880 And it's got two alphas, two betas. 13 00:00:46,880 --> 00:00:52,520 The beta subunit of hemoglobin is encoded by the human beta 14 00:00:52,520 --> 00:00:53,820 globin gene. 15 00:00:53,820 --> 00:00:55,245 That's the nomenclature here. 16 00:00:55,245 --> 00:00:56,870 All right. 17 00:00:56,870 --> 00:00:58,120 Let's clone beta globin. 18 00:01:01,150 --> 00:01:01,670 Same deal. 19 00:01:01,670 --> 00:01:04,709 How are we going to do it? 20 00:01:04,709 --> 00:01:05,959 Any takers? 21 00:01:09,120 --> 00:01:09,800 What should we start with? 22 00:01:09,800 --> 00:01:10,210 Yes? 23 00:01:10,210 --> 00:01:12,436 AUDIENCE: Find a restriction enzyme? 24 00:01:12,436 --> 00:01:14,080 ERIC LANDER: Find a restriction enzyme. 25 00:01:14,080 --> 00:01:16,890 I'll go to catalogue and try EcoRI today. 26 00:01:16,890 --> 00:01:17,800 OK? 27 00:01:17,800 --> 00:01:20,158 Now what? 28 00:01:20,158 --> 00:01:25,078 AUDIENCE: Add it to a bunch of [INAUDIBLE]. 29 00:01:25,078 --> 00:01:26,740 ERIC LANDER: So I'm going to start with human DNA? 30 00:01:26,740 --> 00:01:28,945 AUDIENCE: Yes. 31 00:01:28,945 --> 00:01:31,380 ERIC LANDER: Yes is a good answer. 32 00:01:31,380 --> 00:01:32,050 Yes. 33 00:01:32,050 --> 00:01:33,990 I'm going to start with human DNA. 34 00:01:33,990 --> 00:01:34,370 Good. 35 00:01:34,370 --> 00:01:35,160 Let's get that up there. 36 00:01:35,160 --> 00:01:35,780 We're going to start-- 37 00:01:35,780 --> 00:01:36,290 AUDIENCE: [INAUDIBLE]. 38 00:01:36,290 --> 00:01:36,745 ERIC LANDER: Sorry? 39 00:01:36,745 --> 00:01:38,580 AUDIENCE: [INAUDIBLE]. 40 00:01:38,580 --> 00:01:40,120 ERIC LANDER: So we'll start with human DNA. 41 00:01:44,080 --> 00:01:49,430 There we go, pieces of human DNA cut with EcoRI. 42 00:01:49,430 --> 00:01:50,680 Now, what are we going to do? 43 00:01:53,830 --> 00:01:55,590 Takers? 44 00:01:55,590 --> 00:01:57,590 Let's attach it to a vector. 45 00:01:57,590 --> 00:02:01,560 Actually, wait a second. 46 00:02:01,560 --> 00:02:06,330 This vector here, I told you that there were vectors that 47 00:02:06,330 --> 00:02:07,707 could grow in E. coli. 48 00:02:07,707 --> 00:02:08,930 Did I ever tell you there were vectors that 49 00:02:08,930 --> 00:02:10,180 could grow a yeast? 50 00:02:12,880 --> 00:02:15,800 There are. 51 00:02:15,800 --> 00:02:16,720 And they're in the catalog. 52 00:02:16,720 --> 00:02:17,530 OK. 53 00:02:17,530 --> 00:02:19,000 Yes? 54 00:02:19,000 --> 00:02:19,650 Now, what am I going to do? 55 00:02:19,650 --> 00:02:20,980 I'm going to attach this to a vector. 56 00:02:24,980 --> 00:02:26,900 Now, what do I do? 57 00:02:26,900 --> 00:02:28,490 What vector do I want to attach this to? 58 00:02:31,540 --> 00:02:34,490 Where is it going to grow? 59 00:02:34,490 --> 00:02:35,740 Human cells. 60 00:02:38,960 --> 00:02:40,550 That's interesting. 61 00:02:40,550 --> 00:02:42,365 So one option would be human cells. 62 00:02:42,365 --> 00:02:47,340 So we want to take a vector that grows in human cells. 63 00:02:47,340 --> 00:02:48,590 Ha. 64 00:02:52,880 --> 00:02:53,373 Yep. 65 00:02:53,373 --> 00:02:54,623 AUDIENCE: [INAUDIBLE]. 66 00:02:59,790 --> 00:03:01,790 ERIC LANDER: Then I need a mammalian origin of 67 00:03:01,790 --> 00:03:02,730 replication. 68 00:03:02,730 --> 00:03:05,110 AUDIENCE: [INAUDIBLE]. 69 00:03:05,110 --> 00:03:06,520 ERIC LANDER: And a mammal-- 70 00:03:06,520 --> 00:03:10,380 Well, so it turns out that I can technically do this. 71 00:03:10,380 --> 00:03:12,610 There are now vectors that I can use to 72 00:03:12,610 --> 00:03:15,340 grow in human cells. 73 00:03:15,340 --> 00:03:17,820 It's a lot harder to do. 74 00:03:17,820 --> 00:03:22,680 So let's try doing it with a microbe first, but then we'll 75 00:03:22,680 --> 00:03:24,950 come back and we'll do it with human. 76 00:03:24,950 --> 00:03:26,220 So try doing this in a microbe. 77 00:03:26,220 --> 00:03:28,050 What have you got for a microbe? 78 00:03:28,050 --> 00:03:29,300 How do we do this in a microbe? 79 00:03:35,300 --> 00:03:38,710 Well, let's try E. coli for the sake of argument. 80 00:03:38,710 --> 00:03:42,480 But we could also try, keep in mind your human for a second. 81 00:03:42,480 --> 00:03:45,450 We do this, we do this, we get a plate. 82 00:03:45,450 --> 00:03:49,070 One of these guys is going to have alpha globin, one of them 83 00:03:49,070 --> 00:03:50,500 will have collagen. 84 00:03:50,500 --> 00:03:52,910 That guy here has beta globin. 85 00:03:52,910 --> 00:03:55,770 How are we going to recognize it? 86 00:03:55,770 --> 00:03:57,020 This is tough. 87 00:03:59,400 --> 00:04:00,490 What were you going to do for the human? 88 00:04:00,490 --> 00:04:02,194 AUDIENCE: No idea. 89 00:04:02,194 --> 00:04:03,405 ERIC LANDER: But you were going to get it 90 00:04:03,405 --> 00:04:04,390 into a human cell. 91 00:04:04,390 --> 00:04:04,890 AUDIENCE: Yeah. 92 00:04:04,890 --> 00:04:06,680 ERIC LANDER: Were you going to try to do complementation? 93 00:04:06,680 --> 00:04:07,886 AUDIENCE: Maybe. 94 00:04:07,886 --> 00:04:08,400 ERIC LANDER: Yeah. 95 00:04:08,400 --> 00:04:10,440 The problem doing complementation is most human 96 00:04:10,440 --> 00:04:11,660 cells don't need beta globin. 97 00:04:11,660 --> 00:04:12,900 What's beta globin good for? 98 00:04:12,900 --> 00:04:15,375 AUDIENCE: [INAUDIBLE]. 99 00:04:15,375 --> 00:04:17,472 ERIC LANDER: What's hemoglobin good for? 100 00:04:17,472 --> 00:04:18,316 AUDIENCE: [INAUDIBLE]. 101 00:04:18,316 --> 00:04:20,790 ERIC LANDER: It's in your red blood cells to transport 102 00:04:20,790 --> 00:04:22,500 oxygen around. 103 00:04:22,500 --> 00:04:24,940 You think individual cells care? 104 00:04:24,940 --> 00:04:26,840 Nah. 105 00:04:26,840 --> 00:04:29,130 Your marrow might, but only in C2. 106 00:04:29,130 --> 00:04:31,680 The problem with doing it in a human cell initially is, if we 107 00:04:31,680 --> 00:04:33,920 tried the same exact trick and we tried a beta 108 00:04:33,920 --> 00:04:36,580 globin-deficient human cell, and we're going to try to 109 00:04:36,580 --> 00:04:39,810 complement with a beta globin, the problem is we don't have 110 00:04:39,810 --> 00:04:43,480 any conditions under which that cell cares. 111 00:04:43,480 --> 00:04:45,230 But we'll come back and even do more because we could do 112 00:04:45,230 --> 00:04:45,960 things here. 113 00:04:45,960 --> 00:04:46,970 So now what are we going to do? 114 00:04:46,970 --> 00:04:48,950 How are we going to see where our beta globin is? 115 00:04:48,950 --> 00:04:50,700 AUDIENCE: [INAUDIBLE]. 116 00:04:50,700 --> 00:04:53,800 ERIC LANDER: Make the protein. 117 00:04:53,800 --> 00:04:55,100 So we're going to persuade this E. 118 00:04:55,100 --> 00:04:56,570 coli to make the protein. 119 00:04:56,570 --> 00:04:58,271 And how are we going to recognize the protein? 120 00:04:58,271 --> 00:04:59,955 AUDIENCE: [INAUDIBLE]. 121 00:04:59,955 --> 00:05:03,430 ERIC LANDER: So how can we recognize a protein? 122 00:05:03,430 --> 00:05:05,705 AUDIENCE: Function. 123 00:05:05,705 --> 00:05:06,590 ERIC LANDER: Sorry? 124 00:05:06,590 --> 00:05:07,536 AUDIENCE: Function. 125 00:05:07,536 --> 00:05:09,120 ERIC LANDER: Function. 126 00:05:09,120 --> 00:05:10,880 The problem is we didn't really have a function to 127 00:05:10,880 --> 00:05:12,130 complement. 128 00:05:14,020 --> 00:05:15,490 Electrophoresis. 129 00:05:15,490 --> 00:05:20,110 We could actually take every colony on the plate, grow it 130 00:05:20,110 --> 00:05:24,760 up, purify the proteins, and see if there was a protein 131 00:05:24,760 --> 00:05:28,410 band at just the right place for beta globin. 132 00:05:28,410 --> 00:05:31,590 We'd have to look at about a million colonies to do it. 133 00:05:31,590 --> 00:05:33,550 Maybe a little more, actually more because you know, 134 00:05:33,550 --> 00:05:35,710 statistics and all, 10 million colonies. 135 00:05:35,710 --> 00:05:38,640 And the issue there is graduate students tend to, you 136 00:05:38,640 --> 00:05:41,480 can check with the TAs, but on the whole, tend to rebel at 137 00:05:41,480 --> 00:05:47,530 the thought of prepping protein from 10 million 138 00:05:47,530 --> 00:05:49,030 separate cells. 139 00:05:49,030 --> 00:05:51,780 How else can we tell if our cells are making beta globin? 140 00:05:54,720 --> 00:05:56,680 AUDIENCE: You add a marker to it? 141 00:05:56,680 --> 00:05:59,850 ERIC LANDER: Something that sticks to beta globin. 142 00:05:59,850 --> 00:06:03,480 Turns out that antibodies are really good for this. 143 00:06:03,480 --> 00:06:06,590 If you take beta globin and you inject a mouse with beta 144 00:06:06,590 --> 00:06:08,835 globin, it'll make an immune reaction against it. 145 00:06:08,835 --> 00:06:10,960 And we'll learn more about the immune system in the course, 146 00:06:10,960 --> 00:06:12,840 but it will actually make antibodies that 147 00:06:12,840 --> 00:06:14,540 bind to beta globin. 148 00:06:14,540 --> 00:06:17,030 So suppose I told you there were antibodies that actually 149 00:06:17,030 --> 00:06:18,280 bind to beta globin? 150 00:06:21,940 --> 00:06:22,900 Now, what can I do? 151 00:06:22,900 --> 00:06:24,235 AUDIENCE: [INAUDIBLE]. 152 00:06:24,235 --> 00:06:26,550 ERIC LANDER: I'll make it a fluorescent antibody, sure. 153 00:06:26,550 --> 00:06:28,780 You can have a fluorescent antibody if you want. 154 00:06:28,780 --> 00:06:30,320 Fluorescent antibody that knows how to 155 00:06:30,320 --> 00:06:31,780 bind to beta globin. 156 00:06:31,780 --> 00:06:33,940 Now, how do I find my cell? 157 00:06:33,940 --> 00:06:35,660 AUDIENCE: Colonies. 158 00:06:35,660 --> 00:06:36,900 ERIC LANDER: Take my colonies. 159 00:06:36,900 --> 00:06:37,410 Do what to them? 160 00:06:37,410 --> 00:06:38,874 AUDIENCE: Add antibodies. 161 00:06:38,874 --> 00:06:41,314 And look for the colony that had antibodies-- 162 00:06:41,314 --> 00:06:42,630 ERIC LANDER: Stuck to it. 163 00:06:42,630 --> 00:06:44,780 I just wash antibodies over my colonies and 164 00:06:44,780 --> 00:06:46,470 see where it sticks. 165 00:06:46,470 --> 00:06:49,810 Now, the only details are this is on an agar plate, and it's 166 00:06:49,810 --> 00:06:51,520 really messy to do that. 167 00:06:51,520 --> 00:06:55,010 So just technical details of pulling that off are I 168 00:06:55,010 --> 00:06:57,970 actually grew these on a little piece of filter paper. 169 00:06:57,970 --> 00:07:00,330 So I put a piece of filter paper down. 170 00:07:00,330 --> 00:07:02,490 I grow the colonies on filter paper getting their little 171 00:07:02,490 --> 00:07:03,680 nutrients through there. 172 00:07:03,680 --> 00:07:06,350 I take off my piece of filter paper that has my colonies 173 00:07:06,350 --> 00:07:07,490 growing on it. 174 00:07:07,490 --> 00:07:10,780 I need to crack open all of those cells, and it turns out 175 00:07:10,780 --> 00:07:13,200 I can do that by a chemical treatment that will crack open 176 00:07:13,200 --> 00:07:15,480 all the cells and leave their protein 177 00:07:15,480 --> 00:07:16,960 contents just spewed out. 178 00:07:16,960 --> 00:07:19,570 They spew out their guts right here, each cell. 179 00:07:23,090 --> 00:07:26,190 And then I wash my antibody over it, and my 180 00:07:26,190 --> 00:07:30,550 fluorescently-labeled antibody sticks to that guy telling me 181 00:07:30,550 --> 00:07:33,090 that beta globin was right there. 182 00:07:33,090 --> 00:07:34,340 Any questions? 183 00:07:37,200 --> 00:07:37,835 I did it. 184 00:07:37,835 --> 00:07:39,085 You did it. 185 00:07:41,530 --> 00:07:42,780 Except there's a problem. 186 00:07:45,390 --> 00:07:46,568 What was your problem? 187 00:07:46,568 --> 00:07:49,774 AUDIENCE: Is it possible that the antibody won't stick? 188 00:07:49,774 --> 00:07:51,330 ERIC LANDER: It's a wonderful antibody. 189 00:07:51,330 --> 00:07:52,665 It's a perfectly specific antibody. 190 00:07:52,665 --> 00:07:54,230 The antibody is totally perfect. 191 00:07:54,230 --> 00:07:57,480 It only sticks to beta globin. 192 00:07:57,480 --> 00:07:59,010 Does the bacteria want to make beta globin? 193 00:08:02,110 --> 00:08:05,050 You're throwing this stuff in and if any bacteria actually 194 00:08:05,050 --> 00:08:08,210 did make you beta globin, this would be working perfectly. 195 00:08:08,210 --> 00:08:11,800 The little problem is, will a piece of human DNA thrown into 196 00:08:11,800 --> 00:08:14,890 E. coli make beta globin? 197 00:08:14,890 --> 00:08:16,120 No. 198 00:08:16,120 --> 00:08:17,280 Why not? 199 00:08:17,280 --> 00:08:19,220 Well, let's go back to our understanding 200 00:08:19,220 --> 00:08:20,470 of how genes work. 201 00:08:23,920 --> 00:08:29,210 The beta globin gene is a locus that has a human 202 00:08:29,210 --> 00:08:36,940 promoter which then goes and makes a transcript. 203 00:08:36,940 --> 00:08:41,809 That transcript has multiple exons. 204 00:08:41,809 --> 00:08:45,660 Those exons go into the transcript and are spliced 205 00:08:45,660 --> 00:08:49,490 together to make a mature message. 206 00:08:49,490 --> 00:08:52,830 Here is the mature message, which gets translated. 207 00:08:52,830 --> 00:08:57,480 We have splicing, and then this gets translated. 208 00:08:57,480 --> 00:09:00,070 The spliced product gets translated. 209 00:09:00,070 --> 00:09:03,280 And what's the problem asking E. coli to do this? 210 00:09:03,280 --> 00:09:04,106 AUDIENCE: It doesn't do splicing. 211 00:09:04,106 --> 00:09:07,010 ERIC LANDER: It doesn't do splicing. 212 00:09:07,010 --> 00:09:10,070 And in fact, if you told me we'll use yeast instead, it 213 00:09:10,070 --> 00:09:13,390 turns out yeast does a very bad job in splicing human 214 00:09:13,390 --> 00:09:15,220 introns also. 215 00:09:15,220 --> 00:09:17,405 And E. coli doesn't recognize human promoters. 216 00:09:20,490 --> 00:09:21,740 So this doesn't work very well. 217 00:09:24,760 --> 00:09:26,480 Any takers on what we're going to do about this? 218 00:09:26,480 --> 00:09:27,630 How do we fix this problem? 219 00:09:27,630 --> 00:09:30,310 AUDIENCE: [INAUDIBLE]. 220 00:09:30,310 --> 00:09:32,350 ERIC LANDER: Ah. 221 00:09:32,350 --> 00:09:36,420 So one solution would be teach E. coli how to splice DNA. 222 00:09:36,420 --> 00:09:39,030 That's hard. 223 00:09:39,030 --> 00:09:40,930 Start with cDNA. 224 00:09:40,930 --> 00:09:41,870 That's nice. 225 00:09:41,870 --> 00:09:49,300 So let's take our messenger RNA, mRNA, and let's now 226 00:09:49,300 --> 00:09:51,920 convert it into cDNA. 227 00:09:51,920 --> 00:09:53,170 What is cDNA? 228 00:09:55,320 --> 00:09:59,920 cDNA remember, we can copy RNA into DNA. 229 00:09:59,920 --> 00:10:02,340 What enzyme copies RNA into DNA? 230 00:10:02,340 --> 00:10:04,090 We learned about it when we studied 231 00:10:04,090 --> 00:10:05,390 replication and all that. 232 00:10:05,390 --> 00:10:06,510 AUDIENCE: Reverse transcriptase. 233 00:10:06,510 --> 00:10:08,750 ERIC LANDER: Reverse transcriptase. 234 00:10:08,750 --> 00:10:12,550 So if I can get my hands on some reverse transcriptase, I 235 00:10:12,550 --> 00:10:16,410 would be able to add reverse transcriptase to mRNAs from a 236 00:10:16,410 --> 00:10:18,240 human cell and make cDNA. 237 00:10:18,240 --> 00:10:20,600 And where do I get reversed transcriptase? 238 00:10:20,600 --> 00:10:21,980 It's in the catalog. 239 00:10:21,980 --> 00:10:27,880 So I now make cDNA and instead, I put 240 00:10:27,880 --> 00:10:32,000 the cDNA in the vector. 241 00:10:32,000 --> 00:10:33,380 I do this, of course, for many, many, 242 00:10:33,380 --> 00:10:34,600 many different RNAs. 243 00:10:34,600 --> 00:10:37,900 I take total RNA from the human cell, I convert all the 244 00:10:37,900 --> 00:10:40,780 total RNA all at once into different cDNAs. 245 00:10:40,780 --> 00:10:44,720 Those different cDNAs get put into the vector, and I now 246 00:10:44,720 --> 00:10:47,680 have what's called a cDNA library. 247 00:10:47,680 --> 00:10:49,760 So now I have a cDNA library. 248 00:10:59,780 --> 00:11:03,300 And now each of these guys has not a piece of human genomic 249 00:11:03,300 --> 00:11:09,110 DNA, but a piece of human cDNA copied back from a message. 250 00:11:09,110 --> 00:11:11,430 One of those guys has beta globin. 251 00:11:11,430 --> 00:11:13,240 Now will E. coli produce beta globin? 252 00:11:15,850 --> 00:11:16,280 No. 253 00:11:16,280 --> 00:11:17,160 Because 254 00:11:17,160 --> 00:11:17,946 AUDIENCE: Promoter. 255 00:11:17,946 --> 00:11:18,940 ERIC LANDER: Promoter. 256 00:11:18,940 --> 00:11:22,000 It doesn't have a promoter that it recognizes. 257 00:11:22,000 --> 00:11:22,945 What are we going to do we do about that? 258 00:11:22,945 --> 00:11:24,590 AUDIENCE: Add one in the vector. 259 00:11:24,590 --> 00:11:25,540 ERIC LANDER: Add one in the vector. 260 00:11:25,540 --> 00:11:27,045 Give it a promoter in the vector. 261 00:11:27,045 --> 00:11:27,190 Good. 262 00:11:27,190 --> 00:11:30,130 You guys are getting into the engineering of this. 263 00:11:30,130 --> 00:11:33,755 Let's say that the vector has an E. coli promoter. 264 00:11:37,860 --> 00:11:42,470 Now, each of these cells will actually make that mRNA. 265 00:11:42,470 --> 00:11:46,740 That mRNA is spliced, and it will, in 266 00:11:46,740 --> 00:11:49,160 fact, produce an mRNA. 267 00:11:49,160 --> 00:11:54,570 The cell will translate it if we do this right, and is the 268 00:11:54,570 --> 00:11:57,810 genetic code the same in E. coli in human? 269 00:11:57,810 --> 00:11:59,260 Yup. 270 00:11:59,260 --> 00:12:04,030 So we can actually make, you could make beta globin. 271 00:12:04,030 --> 00:12:09,070 Now, we wash over our antibody, it recognizes which 272 00:12:09,070 --> 00:12:12,800 cell is making beta globin, and it sticks. 273 00:12:12,800 --> 00:12:15,110 There's beta globin. 274 00:12:15,110 --> 00:12:17,466 It works. 275 00:12:17,466 --> 00:12:20,140 Oh, by the way, what have we also just invented here? 276 00:12:22,730 --> 00:12:25,430 We've invented a bacterial cell that's able to produce a 277 00:12:25,430 --> 00:12:28,050 human protein. 278 00:12:28,050 --> 00:12:29,790 What if instead of beta globin, we put in 279 00:12:29,790 --> 00:12:32,790 the gene for insulin? 280 00:12:32,790 --> 00:12:34,040 What would we have invented? 281 00:12:36,600 --> 00:12:40,370 A way to produce insulin without having to purify it 282 00:12:40,370 --> 00:12:44,800 from the pancreases of animals, which is how we used 283 00:12:44,800 --> 00:12:48,590 to have to treat juvenile diabetics. 284 00:12:48,590 --> 00:12:53,130 But instead, if we put the insulin gene in, and we have 285 00:12:53,130 --> 00:12:56,000 an E. coli promoter, let's say, in theory, 286 00:12:56,000 --> 00:12:57,590 we can make us insulin. 287 00:12:57,590 --> 00:13:00,050 And what would we have invented? 288 00:13:00,050 --> 00:13:02,570 The biotechnology industry. 289 00:13:02,570 --> 00:13:05,110 That's basically the start to the biotechnology industry. 290 00:13:05,110 --> 00:13:07,790 One of the first things that was done was cloning the 291 00:13:07,790 --> 00:13:12,330 insulin gene to make recombinantly-produced insulin 292 00:13:12,330 --> 00:13:15,040 by just the method you guys invented today. 293 00:13:15,040 --> 00:13:17,330 It's just unfortunately others invented them first, so you 294 00:13:17,330 --> 00:13:19,930 don't get a patent on it or anything, but all right. 295 00:13:19,930 --> 00:13:26,590 So now we got two ways to clone genes so far, and there 296 00:13:26,590 --> 00:13:27,950 are a lot of ways to clone genes. 297 00:13:27,950 --> 00:13:29,770 I've given you two, later in the course, 298 00:13:29,770 --> 00:13:31,020 I'll give you a third.