1 00:00:06,470 --> 00:00:07,890 PROFESSOR: Good morning, good morning. 2 00:00:16,340 --> 00:00:23,570 So last time, we talked about the most remarkable 3 00:00:23,570 --> 00:00:28,260 biochemical purification procedure ever invented-- 4 00:00:28,260 --> 00:00:29,510 cloning. 5 00:00:31,340 --> 00:00:34,230 You remember the issue with biochemistry. 6 00:00:34,230 --> 00:00:38,120 You're going to grind up a cell, you're going to take the 7 00:00:38,120 --> 00:00:41,210 contents and run it over different kinds of separation 8 00:00:41,210 --> 00:00:46,660 columns or centrifuge it or things, in order to separate 9 00:00:46,660 --> 00:00:48,970 some things from other things. 10 00:00:48,970 --> 00:00:54,300 The problem with purifying a gene that way, away from all 11 00:00:54,300 --> 00:00:57,950 the rest of DNA in a cell, is that the gene has exactly the 12 00:00:57,950 --> 00:01:00,940 same biochemical properties as all the other genes. 13 00:01:00,940 --> 00:01:04,790 How in the world are you going to do it? 14 00:01:04,790 --> 00:01:09,380 The solution was devilishly simple and devilishly complex 15 00:01:09,380 --> 00:01:10,620 at the same time. 16 00:01:10,620 --> 00:01:14,200 All you have to do to purify something is dilute it. 17 00:01:14,200 --> 00:01:16,630 If I take any substance an I add enough 18 00:01:16,630 --> 00:01:19,280 water, it's very dilute. 19 00:01:19,280 --> 00:01:21,966 And in any little drop in any little test tube there will 20 00:01:21,966 --> 00:01:23,470 only be one DNA molecule. 21 00:01:23,470 --> 00:01:25,480 It's now purified. 22 00:01:25,480 --> 00:01:28,130 I could do that for every molecule there is. 23 00:01:28,130 --> 00:01:33,550 The problem is, it's not very useful unless I have a way to 24 00:01:33,550 --> 00:01:40,270 take that molecule and make extra copies of it. 25 00:01:40,270 --> 00:01:43,700 But if I could do that, dilution is purification. 26 00:01:46,300 --> 00:01:51,490 The trick we use initially for making more copies is we ask 27 00:01:51,490 --> 00:01:54,940 E. coli to do it for us. 28 00:01:54,940 --> 00:01:58,680 That, as usual, is the solution to most issues. 29 00:01:58,680 --> 00:02:02,390 Find something in life that already does it. 30 00:02:02,390 --> 00:02:06,360 And so, just to remember what we did, we took total DNA. 31 00:02:06,360 --> 00:02:10,160 Maybe it was yeast DNA, maybe it was human DNA, maybe 32 00:02:10,160 --> 00:02:12,130 it was zebra DNA. 33 00:02:12,130 --> 00:02:14,840 And we cut it up with some restriction enzyme. 34 00:02:17,920 --> 00:02:23,570 Our restriction enzyme we used was EcoR1 And it therefore cut 35 00:02:23,570 --> 00:02:27,560 at the EcoR1 sites, G, A, A, T, T, C. We could have used a 36 00:02:27,560 --> 00:02:30,670 different restriction enzyme. 37 00:02:30,670 --> 00:02:35,850 We then added it to a vector. 38 00:02:35,850 --> 00:02:40,420 The vector was cut open at an EcoR1 site. 39 00:02:40,420 --> 00:02:45,370 The vector had an origin of replication, ORI, and the 40 00:02:45,370 --> 00:02:53,030 vector had a resistance marker, some resistance gene 41 00:02:53,030 --> 00:02:56,740 that made a protein that could break down some antibiotic 42 00:02:56,740 --> 00:02:59,120 found in nature. 43 00:02:59,120 --> 00:03:00,875 We combine these two pieces. 44 00:03:03,990 --> 00:03:06,520 Vector now gets its insert. 45 00:03:06,520 --> 00:03:08,670 We call this vector, we call this insert. 46 00:03:13,100 --> 00:03:16,292 We attach them together using what? 47 00:03:16,292 --> 00:03:16,720 AUDIENCE: Ligase. 48 00:03:16,720 --> 00:03:20,280 PROFESSOR: Ligase, the enzyme that ligates DNA. 49 00:03:20,280 --> 00:03:25,030 We then take this, we transform it into a bacterium. 50 00:03:28,830 --> 00:03:31,860 The scale is obviously off, right? 51 00:03:31,860 --> 00:03:36,750 This DNA plasmid here is tiny compared to this bacteria. 52 00:03:36,750 --> 00:03:39,400 But if I draw it to scale, it won't be very helpful. 53 00:03:39,400 --> 00:03:42,640 So we then transform it in here. 54 00:03:42,640 --> 00:03:44,200 We do that in a test tube. 55 00:03:44,200 --> 00:03:47,310 We treat the bacteria a little roughly so it likes to slurp 56 00:03:47,310 --> 00:03:48,640 up the DNA. 57 00:03:48,640 --> 00:03:51,650 We then plate it on a plate. 58 00:03:51,650 --> 00:03:57,670 And those bacteria that have acquired our plasmid 59 00:03:57,670 --> 00:04:03,800 containing the antibiotic resistance gene are able to 60 00:04:03,800 --> 00:04:09,600 grow on this plate that has antibiotic on it. 61 00:04:09,600 --> 00:04:14,670 And we call this thing a library. 62 00:04:14,670 --> 00:04:16,320 So that's it. 63 00:04:16,320 --> 00:04:17,769 We can make a library. 64 00:04:17,769 --> 00:04:19,329 And we have in effect, then, what I said. 65 00:04:19,329 --> 00:04:23,400 We've deluded the individual molecules out, and each one 66 00:04:23,400 --> 00:04:27,010 went into its own bacteria, and each one got replicated by 67 00:04:27,010 --> 00:04:28,260 that bacteria. 68 00:04:28,260 --> 00:04:30,530 Those plasmids are replicated by the bacteria. 69 00:04:30,530 --> 00:04:33,090 In fact, we choose plasmids that are called multicopy 70 00:04:33,090 --> 00:04:36,320 plasmids, where there's not just one copy but the cell 71 00:04:36,320 --> 00:04:37,980 might make 50 copies of it. 72 00:04:37,980 --> 00:04:42,070 We grow up a whole colony of it and there you go. 73 00:04:42,070 --> 00:04:45,490 We talked about some of the issues with it. 74 00:04:45,490 --> 00:04:47,650 Where do we get the restriction enzymes, the 75 00:04:47,650 --> 00:04:51,830 ligases, the vectors, et cetera? 76 00:04:51,830 --> 00:04:53,440 It's in the catalog, right? 77 00:04:53,440 --> 00:04:55,940 We used to purify them ourselves, but they're all in 78 00:04:55,940 --> 00:04:57,430 the catalog. 79 00:04:57,430 --> 00:05:01,500 So any questions about this? 80 00:05:01,500 --> 00:05:02,350 We had some questions. 81 00:05:02,350 --> 00:05:05,540 I have a question about this. 82 00:05:05,540 --> 00:05:11,860 How come when we add ligase the vector itself just doesn't 83 00:05:11,860 --> 00:05:17,190 close up into a circle without an insert in it? 84 00:05:17,190 --> 00:05:18,520 It might. 85 00:05:18,520 --> 00:05:20,346 What would happen then? 86 00:05:20,346 --> 00:05:21,780 AUDIENCE: You'd just have your original vector. 87 00:05:21,780 --> 00:05:22,890 PROFESSOR: You'd just have your original vector. 88 00:05:22,890 --> 00:05:26,174 And what happens when I transform into the bacteria? 89 00:05:26,174 --> 00:05:27,620 AUDIENCE: It'll still survive. 90 00:05:27,620 --> 00:05:29,050 PROFESSOR: It'll still survive. 91 00:05:29,050 --> 00:05:32,920 So will the unimolecular closure of that circle be more 92 00:05:32,920 --> 00:05:36,120 common than the bimolecular interaction between a vector 93 00:05:36,120 --> 00:05:38,330 and an insert? 94 00:05:38,330 --> 00:05:38,685 AUDIENCE: Probably. 95 00:05:38,685 --> 00:05:40,500 PROFESSOR: Probably, because the two ends of the circle are 96 00:05:40,500 --> 00:05:42,480 pretty close to each other. 97 00:05:42,480 --> 00:05:45,422 So what do I do? 98 00:05:45,422 --> 00:05:46,418 Yeah? 99 00:05:46,418 --> 00:05:47,912 AUDIENCE: Because [INAUDIBLE] 100 00:05:47,912 --> 00:05:49,904 are the same. 101 00:05:49,904 --> 00:05:50,277 [INAUDIBLE] 102 00:05:50,277 --> 00:05:52,643 same restriction enzymes because if they're two 103 00:05:52,643 --> 00:05:54,400 different enzymes so they don't match. 104 00:05:54,400 --> 00:05:54,970 PROFESSOR: Ooh. 105 00:05:54,970 --> 00:05:56,320 That's cute trick. 106 00:05:56,320 --> 00:05:59,620 Two different restriction enzymes so that 107 00:05:59,620 --> 00:06:02,090 they couldn't reclose. 108 00:06:02,090 --> 00:06:04,640 But then my fragments better have those two different 109 00:06:04,640 --> 00:06:07,530 sites, too, and only be able to clone those fragments that 110 00:06:07,530 --> 00:06:08,550 have the two different ones. 111 00:06:08,550 --> 00:06:09,470 But that could work. 112 00:06:09,470 --> 00:06:11,730 So you want a trick for making sure it doesn't reclose. 113 00:06:14,840 --> 00:06:15,780 Any other tricks? 114 00:06:15,780 --> 00:06:17,535 But the problem is, I won't get all the fragments, only 115 00:06:17,535 --> 00:06:20,020 the ones that have, say, an Eco at a Bam site. 116 00:06:20,020 --> 00:06:20,910 But that's-- 117 00:06:20,910 --> 00:06:24,140 So I bring this up not because it's particularly important, 118 00:06:24,140 --> 00:06:27,220 but to tell you the kind of engineering that really does 119 00:06:27,220 --> 00:06:29,220 have to go on in molecular biology. 120 00:06:29,220 --> 00:06:33,080 What happens is when you have your DNA put in here, we have 121 00:06:33,080 --> 00:06:35,590 a sugar-phosphate backbone in both cases. 122 00:06:35,590 --> 00:06:39,430 And if we look up close, one of these sides has the 123 00:06:39,430 --> 00:06:44,210 phosphate, the other has a hydroxyl. 124 00:06:44,210 --> 00:06:46,830 Phosphate, hydroxyl, phosphate, hydroxyl, right? 125 00:06:46,830 --> 00:06:49,640 And ligase comes in and joins that. 126 00:06:52,570 --> 00:06:56,680 So this guy has a phosphate on this strand, and this guy has 127 00:06:56,680 --> 00:06:58,980 a phosphate on that strand, hydroxyl 128 00:06:58,980 --> 00:07:01,630 there, hydroxyl there. 129 00:07:01,630 --> 00:07:03,000 What would happen if I got rid of the 130 00:07:03,000 --> 00:07:06,580 phosphates on the vector? 131 00:07:06,580 --> 00:07:08,570 Could it reclose? 132 00:07:08,570 --> 00:07:09,540 AUDIENCE: No. 133 00:07:09,540 --> 00:07:11,000 PROFESSOR: No. 134 00:07:11,000 --> 00:07:15,770 So if there were an enzyme that removed phosphates, I 135 00:07:15,770 --> 00:07:17,410 could treat my vector first with the 136 00:07:17,410 --> 00:07:19,530 phosphate-removing enzyme. 137 00:07:19,530 --> 00:07:23,420 And now it couldn't possibly reclose on itself. 138 00:07:23,420 --> 00:07:26,180 And is there such an enzyme? 139 00:07:26,180 --> 00:07:28,150 And what's it called? 140 00:07:28,150 --> 00:07:32,710 Phosphatase, because it takes phosphates so easy. 141 00:07:32,710 --> 00:07:34,830 Phosphatase, it takes off the phosphates. 142 00:07:34,830 --> 00:07:37,360 And then it can't reclose anymore. 143 00:07:37,360 --> 00:07:39,900 And where do we get phosphatase? 144 00:07:39,900 --> 00:07:40,700 It's in the catalog. 145 00:07:40,700 --> 00:07:41,740 Exactly. 146 00:07:41,740 --> 00:07:46,220 So now what happens is that the vector 147 00:07:46,220 --> 00:07:47,850 only has an OH here. 148 00:07:50,580 --> 00:07:53,030 What happens to ligase? 149 00:07:53,030 --> 00:07:58,160 When I put an insert in here, ligase can make a covalent 150 00:07:58,160 --> 00:08:00,230 join on this strand. 151 00:08:00,230 --> 00:08:02,170 But it can't actually make a covalent join 152 00:08:02,170 --> 00:08:05,080 on the other strand. 153 00:08:05,080 --> 00:08:07,580 But does it matter? 154 00:08:07,580 --> 00:08:11,260 No, because I've closed up my circle on that strand, and I 155 00:08:11,260 --> 00:08:13,390 close up the circle there on the other strand, and I just 156 00:08:13,390 --> 00:08:14,840 throw it into E. coli. 157 00:08:14,840 --> 00:08:16,510 And you know what happens when it goes into E. coli? 158 00:08:16,510 --> 00:08:18,370 It's got a nick, obviously, on that strand. 159 00:08:18,370 --> 00:08:19,500 It hasn't closed up. 160 00:08:19,500 --> 00:08:23,270 But what does E. coli do when it sees that DNA? 161 00:08:23,270 --> 00:08:24,860 Must be damaged DNA. 162 00:08:24,860 --> 00:08:26,540 I'll fix it. 163 00:08:26,540 --> 00:08:29,380 So E. coli actually does the last little trick of closing 164 00:08:29,380 --> 00:08:31,940 that up for you with its own enzymes for 165 00:08:31,940 --> 00:08:33,630 repairing its own DNA. 166 00:08:33,630 --> 00:08:36,039 I bring this up not because it's crucial that you should 167 00:08:36,039 --> 00:08:38,690 worry about it, but because I want to know that there's a 168 00:08:38,690 --> 00:08:41,490 whole layer of these interesting engineering tricks 169 00:08:41,490 --> 00:08:42,370 that get developed. 170 00:08:42,370 --> 00:08:45,510 Every one of them exploits enzymes that we know. 171 00:08:45,510 --> 00:08:48,610 Every one of them deals with questions like, will my vector 172 00:08:48,610 --> 00:08:51,730 reclose on itself, how do I avoid that? 173 00:08:51,730 --> 00:08:55,230 And there's a vast cooking book of protocols 174 00:08:55,230 --> 00:08:56,610 in molecular biology. 175 00:08:56,610 --> 00:09:00,230 And we constantly are just cribbing from things life does 176 00:09:00,230 --> 00:09:02,180 to make our protocols more and more efficient. 177 00:09:02,180 --> 00:09:04,710 So I bring it up more because it's kind of a cool thing that 178 00:09:04,710 --> 00:09:08,390 all that goes on, and because it helps you remember that 179 00:09:08,390 --> 00:09:10,780 these phosphates are very important to 180 00:09:10,780 --> 00:09:12,780 joining things up. 181 00:09:12,780 --> 00:09:14,075 That's a digression. 182 00:09:14,075 --> 00:09:16,310 Now, let's go to the topic. 183 00:09:16,310 --> 00:09:19,460 How do we actually read the library? 184 00:09:19,460 --> 00:09:20,710 How do we read our library? 185 00:09:23,490 --> 00:09:26,350 How do we use the library, read from the library? 186 00:09:26,350 --> 00:09:34,350 Well let's say we're going to try to find the arginine gene. 187 00:09:34,350 --> 00:09:37,530 We talked about the gene for arginine in yeast. 188 00:09:37,530 --> 00:09:41,210 So I'd like to clone the gene for ARG1. 189 00:09:41,210 --> 00:09:44,370 We found mutants before that were unable to grow without 190 00:09:44,370 --> 00:09:45,880 supplemental arginine. 191 00:09:45,880 --> 00:09:49,070 They somehow had a defect in producing their own origin. 192 00:09:49,070 --> 00:09:49,760 It's a mutant. 193 00:09:49,760 --> 00:09:52,220 I want to find the gene, please. 194 00:09:52,220 --> 00:09:54,500 How do I do it? 195 00:09:54,500 --> 00:09:58,400 We've got to think about what's our insert DNA. 196 00:09:58,400 --> 00:10:01,410 What are our vectors? 197 00:10:01,410 --> 00:10:04,050 What insert DNA should we start with, zebra? 198 00:10:06,920 --> 00:10:08,160 No. 199 00:10:08,160 --> 00:10:10,410 Human? 200 00:10:10,410 --> 00:10:12,370 No. 201 00:10:12,370 --> 00:10:13,110 How about yeast? 202 00:10:13,110 --> 00:10:15,160 We're trying to clone a gene from yeast, right? 203 00:10:15,160 --> 00:10:16,820 So let's start with yeast. 204 00:10:16,820 --> 00:10:17,380 OK. 205 00:10:17,380 --> 00:10:18,780 So we're going to start with yeast DNA. 206 00:10:25,860 --> 00:10:27,320 We're going to cut up yeast DNA. 207 00:10:32,450 --> 00:10:36,330 We're going to attach it to our vector, we're going to 208 00:10:36,330 --> 00:10:41,100 transform it into E. coli, E. coli will 209 00:10:41,100 --> 00:10:43,670 grow up on our plates. 210 00:10:43,670 --> 00:10:46,070 And one of these guys, I happen to know it's that one 211 00:10:46,070 --> 00:10:49,860 there, contains the ARG1 gene. 212 00:10:49,860 --> 00:10:54,320 The problem is I happen to know it, but you don't. 213 00:10:54,320 --> 00:10:56,420 How are you going to find out where the ARG1 gene is? 214 00:11:04,520 --> 00:11:06,110 Any takers? 215 00:11:06,110 --> 00:11:07,050 Yeah? 216 00:11:07,050 --> 00:11:09,948 AUDIENCE: It could be like when you put the gene in make 217 00:11:09,948 --> 00:11:12,700 it flourescent. 218 00:11:12,700 --> 00:11:14,440 PROFESSOR: A fluorescent tag? 219 00:11:14,440 --> 00:11:16,580 So I should just attach the fluorescent 220 00:11:16,580 --> 00:11:18,280 tag to the ARG1 gene? 221 00:11:18,280 --> 00:11:19,160 AUDIENCE: Yes. 222 00:11:19,160 --> 00:11:19,970 PROFESSOR: How do I do that? 223 00:11:19,970 --> 00:11:21,590 All the DNA looks the same in the tube. 224 00:11:21,590 --> 00:11:25,188 How do I know where to attach the fluorescent tag? 225 00:11:25,188 --> 00:11:28,390 AUDIENCE: Maybe you could size it [INAUDIBLE]. 226 00:11:28,390 --> 00:11:30,530 PROFESSOR: There's a lot of pieces of DNA there. 227 00:11:30,530 --> 00:11:32,824 And my eyes are not that good. 228 00:11:32,824 --> 00:11:33,650 AUDIENCE: Separate it? 229 00:11:33,650 --> 00:11:35,960 PROFESSOR: Separate it. 230 00:11:35,960 --> 00:11:37,810 But will I know which one is ARG1? 231 00:11:37,810 --> 00:11:39,480 See, I don't actually know anything about ARG1. 232 00:11:39,480 --> 00:11:40,870 All I know is I made a mutant. 233 00:11:40,870 --> 00:11:43,370 The mutant is unable to grow without arginine. 234 00:11:43,370 --> 00:11:45,800 I haven't got a clue what that gene is. 235 00:11:45,800 --> 00:11:48,040 I don't know what it encodes, I don't know how big it is, I 236 00:11:48,040 --> 00:11:49,550 don't know nothing. 237 00:11:49,550 --> 00:11:54,430 All I know is that whatever it is, it's a gene which when 238 00:11:54,430 --> 00:11:58,176 mutated prevents you from growing without arginine. 239 00:11:58,176 --> 00:12:01,032 AUDIENCE: Could you plate all of your colonies onto a-- 240 00:12:01,032 --> 00:12:03,412 could you put [INAUDIBLE]? 241 00:12:03,412 --> 00:12:04,510 PROFESSOR: Minimal medium. 242 00:12:04,510 --> 00:12:07,740 What if I plate on minimal medium? 243 00:12:07,740 --> 00:12:08,740 Now what? 244 00:12:08,740 --> 00:12:09,665 What are you hoping for? 245 00:12:09,665 --> 00:12:14,560 AUDIENCE: The one that has the ARG1 gene will not grow. 246 00:12:14,560 --> 00:12:17,760 PROFESSOR: The one that has the ARG1 gene 247 00:12:17,760 --> 00:12:20,180 won't be able to grow-- 248 00:12:20,180 --> 00:12:21,170 oh wait, yeah. 249 00:12:21,170 --> 00:12:22,810 But something like that. 250 00:12:22,810 --> 00:12:23,850 Let's work it through. 251 00:12:23,850 --> 00:12:24,830 We've got my idea here. 252 00:12:24,830 --> 00:12:26,992 What are we going to do with it? 253 00:12:26,992 --> 00:12:28,242 AUDIENCE: [INAUDIBLE] 254 00:12:32,460 --> 00:12:36,830 PROFESSOR: I've got a working ARG1 Mutate ARG1 afterwards? 255 00:12:36,830 --> 00:12:39,490 AUDIENCE: [INAUDIBLE] 256 00:12:39,490 --> 00:12:40,200 PROFESSOR: OK. 257 00:12:40,200 --> 00:12:41,350 How will that work? 258 00:12:41,350 --> 00:12:43,170 I'm open for-- 259 00:12:43,170 --> 00:12:44,340 got an idea here? 260 00:12:44,340 --> 00:12:46,116 AUDIENCE: If you have your different colonies 261 00:12:46,116 --> 00:12:50,456 [INAUDIBLE], you could have a secondary plate them all over 262 00:12:50,456 --> 00:12:51,440 to one of middle medium. 263 00:12:51,440 --> 00:12:56,160 The ones that die are the ones that already [INAUDIBLE]-- 264 00:12:56,160 --> 00:12:58,850 PROFESSOR: So guys, I have a concern. 265 00:12:58,850 --> 00:13:02,190 I'm just transferring this into E. coli. 266 00:13:02,190 --> 00:13:04,490 E.coli grows just fine without arginine. 267 00:13:07,630 --> 00:13:09,800 I mean, I'm going to take this yeast DNA. 268 00:13:09,800 --> 00:13:10,880 I'm going to put it in E. coli. 269 00:13:10,880 --> 00:13:13,290 E. coli was kind enough to grow it for me. 270 00:13:13,290 --> 00:13:17,800 But frankly, E. coli doesn't need this ARG1 gene. 271 00:13:17,800 --> 00:13:19,200 E. coli can grow without arginine. 272 00:13:19,200 --> 00:13:21,520 I can plate this with and without arginine, E. coli 273 00:13:21,520 --> 00:13:22,930 grows just fine. 274 00:13:22,930 --> 00:13:24,150 But you're on to something. 275 00:13:24,150 --> 00:13:27,140 You're onto the idea that somehow, the only thing we 276 00:13:27,140 --> 00:13:34,120 know about ARG1 is that the functional, wild-type copy of 277 00:13:34,120 --> 00:13:40,570 that gene confers an ability to grow without arginine. 278 00:13:40,570 --> 00:13:44,110 And who does it confer it on? 279 00:13:44,110 --> 00:13:46,820 And what kind of yeast? 280 00:13:46,820 --> 00:13:51,420 Haploid mutant yeast. 281 00:13:51,420 --> 00:13:52,570 Ah. 282 00:13:52,570 --> 00:13:57,310 So suppose I put a working copy of ARG1, a good copy, a 283 00:13:57,310 --> 00:14:01,770 wild-type copy, into a mutant yeast. 284 00:14:01,770 --> 00:14:04,080 Now what would happen to that mutant yeast? 285 00:14:08,270 --> 00:14:08,780 What would happen? 286 00:14:08,780 --> 00:14:12,260 The mutant yeast before, could it grow without arginine? 287 00:14:12,260 --> 00:14:13,100 No. 288 00:14:13,100 --> 00:14:14,990 If I put in a working copy of the ARG1 289 00:14:14,990 --> 00:14:17,650 gene, what will happen? 290 00:14:17,650 --> 00:14:19,320 It grows. 291 00:14:19,320 --> 00:14:20,720 Now let's design a scheme. 292 00:14:25,820 --> 00:14:28,040 Do I want to use E. coli at all? 293 00:14:30,570 --> 00:14:31,870 No. 294 00:14:31,870 --> 00:14:34,226 What do I want to use? 295 00:14:34,226 --> 00:14:35,670 I want to use a yeast. 296 00:14:35,670 --> 00:14:40,330 So let's get rid of E. coli and let's instead use yeast. 297 00:14:40,330 --> 00:14:44,140 And which yeast should we use, wild-type or mutant? 298 00:14:44,140 --> 00:14:45,280 Mutant yeast. 299 00:14:45,280 --> 00:14:47,280 What mutation? 300 00:14:47,280 --> 00:14:50,210 ARG1 mutant yeast. 301 00:14:50,210 --> 00:14:53,000 ARG1 minus yeast. 302 00:14:53,000 --> 00:14:57,740 Now, if I plate ARG1 minus yeast on minimal 303 00:14:57,740 --> 00:15:00,330 medium, what happens? 304 00:15:00,330 --> 00:15:01,830 It doesn't grow. 305 00:15:01,830 --> 00:15:04,520 It dies. 306 00:15:04,520 --> 00:15:08,585 What DNA should I be putting in? 307 00:15:08,585 --> 00:15:11,070 Yeast DNA. 308 00:15:11,070 --> 00:15:13,370 Mutant or wild type? 309 00:15:13,370 --> 00:15:16,110 Why wild type? 310 00:15:16,110 --> 00:15:19,070 Because it'll have a working copy of ARG1. 311 00:15:19,070 --> 00:15:22,600 So I want yeast, wild type. 312 00:15:22,600 --> 00:15:24,410 Now what happens? 313 00:15:24,410 --> 00:15:27,680 One of these guys, and only one of these guys here, this 314 00:15:27,680 --> 00:15:31,970 one, has a ARG1 gene. 315 00:15:31,970 --> 00:15:33,840 That's ARG1. 316 00:15:33,840 --> 00:15:40,720 When it goes in, that plasmid has the ARG1 plus gene, 317 00:15:40,720 --> 00:15:44,430 whereas other plasmids don't. 318 00:15:44,430 --> 00:15:47,550 That cell that inherits that gene there, that gets that 319 00:15:47,550 --> 00:15:49,320 gene, is not green. 320 00:15:49,320 --> 00:15:51,050 I just drew it green for you. 321 00:15:51,050 --> 00:15:52,850 But it has the ARG1 gene. 322 00:15:52,850 --> 00:15:54,950 And when I plate this on minimal medium, what's 323 00:15:54,950 --> 00:15:56,650 distinctive about it? 324 00:15:56,650 --> 00:15:58,280 It grows. 325 00:15:58,280 --> 00:16:01,470 That's how you can clone the ARG1 gene. 326 00:16:01,470 --> 00:16:03,910 You clone it by the only thing you know about it. 327 00:16:03,910 --> 00:16:06,520 Namely, it confers a function. 328 00:16:06,520 --> 00:16:10,880 This is called cloning by function. 329 00:16:10,880 --> 00:16:22,290 Or, what did we do when we crossed two mutants together 330 00:16:22,290 --> 00:16:26,500 to see if things were in different genes? 331 00:16:26,500 --> 00:16:29,990 It was a test of complementation. 332 00:16:29,990 --> 00:16:34,040 Really what we're asking is, is there a plasmid that 333 00:16:34,040 --> 00:16:38,400 complements the defect? 334 00:16:38,400 --> 00:16:44,180 In effect what's happening is in this cell, right here, we 335 00:16:44,180 --> 00:16:45,760 have a yeast cell. 336 00:16:45,760 --> 00:16:50,470 And the yeast cell has a defect in its ARG1. 337 00:16:50,470 --> 00:16:55,760 But the plasmid has a working ARG1. 338 00:16:55,760 --> 00:17:00,140 So for that one gene, this cell could be thought of maybe 339 00:17:00,140 --> 00:17:03,700 a little bit like a diploid, just at that one gene. 340 00:17:03,700 --> 00:17:07,569 And we've done a complementation, just a teeny 341 00:17:07,569 --> 00:17:09,780 little complementation for one gene. 342 00:17:09,780 --> 00:17:13,920 And we could call this cloning complementation. 343 00:17:19,930 --> 00:17:21,405 It's essentially cloning by function. 344 00:17:30,380 --> 00:17:32,190 Any questions? 345 00:17:32,190 --> 00:17:33,884 Yes? 346 00:17:33,884 --> 00:17:36,369 AUDIENCE: [INAUDIBLE] 347 00:17:36,369 --> 00:17:40,345 --they all have functioning [INAUDIBLE]? 348 00:17:40,345 --> 00:17:41,836 PROFESSOR: Yep. 349 00:17:41,836 --> 00:17:44,840 AUDIENCE: So why does that then have a function? 350 00:17:44,840 --> 00:17:45,520 PROFESSOR: Oh, oh. 351 00:17:45,520 --> 00:17:48,800 You see, the yeast genome has about 4,000 different genes. 352 00:17:48,800 --> 00:17:50,910 I chop it up with my EcoR1. 353 00:17:50,910 --> 00:17:56,090 Some plasmids get ARG1 but most of them get leucine 2 or 354 00:17:56,090 --> 00:17:57,200 [INAUDIBLE] 355 00:17:57,200 --> 00:17:58,460 or other things. 356 00:17:58,460 --> 00:18:02,040 And each yeast cell in my library only picks up a 357 00:18:02,040 --> 00:18:05,160 plasmid with one chunk of DNA, one gene. 358 00:18:05,160 --> 00:18:09,180 So it turns out that the yeast cells in my library, each one 359 00:18:09,180 --> 00:18:12,080 has one of thousands of alternative possibilities. 360 00:18:12,080 --> 00:18:14,480 And it's just the guy who gets ARG1 who grows. 361 00:18:14,480 --> 00:18:17,360 AUDIENCE: But you're saying that the yeast [INAUDIBLE] 362 00:18:17,360 --> 00:18:18,320 plasmid. 363 00:18:18,320 --> 00:18:21,210 All code for-- 364 00:18:21,210 --> 00:18:21,780 PROFESSOR: That's right. 365 00:18:21,780 --> 00:18:24,570 AUDIENCE: But I don't get why you would end up with 366 00:18:24,570 --> 00:18:26,500 [INAUDIBLE]. 367 00:18:26,500 --> 00:18:27,250 PROFESSOR: What do I--? 368 00:18:27,250 --> 00:18:29,162 AUDIENCE: Why do you end up with a strain 369 00:18:29,162 --> 00:18:30,600 that has ARG1 in it? 370 00:18:30,600 --> 00:18:31,090 PROFESSOR: Oh. 371 00:18:31,090 --> 00:18:33,830 ARG1 is the ARG1 working copy. 372 00:18:33,830 --> 00:18:37,350 In the yeast, I'm talking about this yeast here has the 373 00:18:37,350 --> 00:18:41,160 working copy of ARG1, ARG1 plus. 374 00:18:41,160 --> 00:18:44,200 So this guy has an ARG1 plus. 375 00:18:44,200 --> 00:18:46,610 It also has lots of other genes. 376 00:18:46,610 --> 00:18:49,330 Each of these plasmids gets one gene. 377 00:18:49,330 --> 00:18:52,150 Some of them get an ARG plus. 378 00:18:52,150 --> 00:18:55,500 Some of them happen to get a leucine gene or some other 379 00:18:55,500 --> 00:18:56,690 gene that's irrelevant. 380 00:18:56,690 --> 00:19:01,544 And the plasmids that contain the working copy of the gene, 381 00:19:01,544 --> 00:19:04,600 they, when they go into the cell, give the cell the 382 00:19:04,600 --> 00:19:05,930 ability to grow. 383 00:19:05,930 --> 00:19:07,005 So that's why. 384 00:19:07,005 --> 00:19:07,710 All right. 385 00:19:07,710 --> 00:19:09,980 So that's how we get a gene by function.