1 00:00:16,750 --> 00:00:20,470 ADAM MARTIN: All right, so we're going to switch gears again 2 00:00:20,470 --> 00:00:25,060 today, and we're going to move off of kind of pure genetics 3 00:00:25,060 --> 00:00:28,150 and start to talk about molecular genetics. 4 00:00:28,150 --> 00:00:32,650 And I want to start with the concept of-- let's say 5 00:00:32,650 --> 00:00:36,490 you want to identify a piece of DNA, 6 00:00:36,490 --> 00:00:40,210 purify it, and propagate it so that you have it 7 00:00:40,210 --> 00:00:43,000 for future use. 8 00:00:43,000 --> 00:00:49,360 And so the process of doing this is known as cloning. 9 00:00:49,360 --> 00:00:52,750 And it's the process of, if you will, 10 00:00:52,750 --> 00:00:59,140 purifying and propagating a piece of DNA in an organism. 11 00:01:07,900 --> 00:01:10,600 So sort of the goal for this lecture 12 00:01:10,600 --> 00:01:14,530 is for you to know how if you wanted to, let's say, 13 00:01:14,530 --> 00:01:16,690 identify a piece of DNA-- 14 00:01:16,690 --> 00:01:18,850 maybe you're interested in the piece of DNA 15 00:01:18,850 --> 00:01:20,860 that contains a given gene. 16 00:01:20,860 --> 00:01:26,170 How would you go about getting that DNA in a state that can be 17 00:01:26,170 --> 00:01:29,050 propagated sort of on and on? 18 00:01:29,050 --> 00:01:31,930 And how can you identify the piece of DNA you want? 19 00:01:34,510 --> 00:01:37,270 And so one tool that we're going to use 20 00:01:37,270 --> 00:01:43,010 is we're going to use an organism bacteria as a tool. 21 00:01:43,010 --> 00:01:48,070 So I'll draw my sample bacteria cell here. 22 00:01:48,070 --> 00:01:51,670 Could be something like E. coli, just some bacterium. 23 00:01:56,620 --> 00:02:03,280 And you'll recall, when I talked about cells a few weeks ago, 24 00:02:03,280 --> 00:02:06,580 bacteria and prokaryotic cells have 25 00:02:06,580 --> 00:02:11,560 a chromosome called the nucleoid that's present inside. 26 00:02:11,560 --> 00:02:13,420 So that's the bacterial chromosome. 27 00:02:16,780 --> 00:02:21,760 But bacteria can also have extra chromosomal pieces of DNA 28 00:02:21,760 --> 00:02:26,308 that are called plasmids that exist in their cytoplasm. 29 00:02:26,308 --> 00:02:27,100 These are plasmids. 30 00:02:30,760 --> 00:02:34,360 And these extra chromosomal pieces of DNA 31 00:02:34,360 --> 00:02:38,440 replicate independently of the chromosome. 32 00:02:38,440 --> 00:02:41,200 And they can persist in the bacterial cell 33 00:02:41,200 --> 00:02:43,390 and be passed on to the daughters 34 00:02:43,390 --> 00:02:48,520 of this bacterial cell as the bacterial cells divide. 35 00:02:48,520 --> 00:02:53,200 So if we focus on an individual plasmid, what a plasmid would 36 00:02:53,200 --> 00:03:01,840 look like, often they have a cassette or a gene that 37 00:03:01,840 --> 00:03:05,260 confers antibiotic resistance. 38 00:03:05,260 --> 00:03:09,340 And that's often the reason that these bacteria are harboring 39 00:03:09,340 --> 00:03:13,510 these plasmids, because it gives them a sort of advantage 40 00:03:13,510 --> 00:03:16,450 if they're exposed to a certain antibiotic 41 00:03:16,450 --> 00:03:19,460 from a predator organism. 42 00:03:19,460 --> 00:03:23,590 So this would confer antibiotic resistance. 43 00:03:27,820 --> 00:03:30,770 One common example is ampicillin resistance, 44 00:03:30,770 --> 00:03:35,380 which I'll abbreviate just amp with an R next to it. 45 00:03:35,380 --> 00:03:40,030 But these plasmids, for them to propagate from bacterial cell 46 00:03:40,030 --> 00:03:42,100 to bacterial cell, they also need 47 00:03:42,100 --> 00:03:44,680 to be able to undergo replication. 48 00:03:44,680 --> 00:03:52,720 So they also have what is known as an origin of replication, 49 00:03:52,720 --> 00:03:57,880 which is often abbreviated ori, which basically allows 50 00:03:57,880 --> 00:04:03,850 this plasmid to be replicated such that copies of the plasma 51 00:04:03,850 --> 00:04:10,390 are passed on to the subsequent generation of bacteria. 52 00:04:10,390 --> 00:04:13,750 And we can take advantage of this plasmid system 53 00:04:13,750 --> 00:04:17,450 in bacteria, because we could take, let's say, 54 00:04:17,450 --> 00:04:20,420 a piece of foreign DNA-- 55 00:04:20,420 --> 00:04:23,980 and this foreign DNA could be of eukaryotic origin. 56 00:04:26,500 --> 00:04:29,620 We could take a piece of eukaryotic DNA 57 00:04:29,620 --> 00:04:31,900 and insert it inside of this plasmid. 58 00:04:34,900 --> 00:04:40,780 And we can basically use the plasma as a sort of platform 59 00:04:40,780 --> 00:04:44,260 or a vector to carry the piece of DNA 60 00:04:44,260 --> 00:04:46,960 that we might be interested in and to use 61 00:04:46,960 --> 00:04:51,370 the bacteria to replicate that DNA and pass it 62 00:04:51,370 --> 00:04:53,530 on to its descendants so that you essentially 63 00:04:53,530 --> 00:04:56,110 have a clone of bacteria, and you 64 00:04:56,110 --> 00:05:01,150 have a clone of this DNA in a given bacterial population. 65 00:05:01,150 --> 00:05:04,210 So, again, this would have an origin and maybe 66 00:05:04,210 --> 00:05:06,550 an ampicillin resistance to it. 67 00:05:09,170 --> 00:05:11,350 So how would you determine whether or not 68 00:05:11,350 --> 00:05:15,416 bacteria have a plasmid in it? 69 00:05:15,416 --> 00:05:17,110 Can you think of an experiment you 70 00:05:17,110 --> 00:05:22,730 could do to determine whether the bacteria has this plasmid? 71 00:05:22,730 --> 00:05:23,410 Stephen? 72 00:05:23,410 --> 00:05:24,868 AUDIENCE: You could add ampicillin, 73 00:05:24,868 --> 00:05:26,555 and it'll survive with the plasmid. 74 00:05:26,555 --> 00:05:27,430 ADAM MARTIN: Exactly. 75 00:05:27,430 --> 00:05:31,360 So what Stephen suggested is that if he 76 00:05:31,360 --> 00:05:34,510 wanted to know whether or not this bacteria had 77 00:05:34,510 --> 00:05:38,950 the plasmid in it, he would add ampicillin to the media. 78 00:05:38,950 --> 00:05:42,190 And if the bacteria doesn't have the plasmid, 79 00:05:42,190 --> 00:05:43,940 it won't be able to grow. 80 00:05:43,940 --> 00:05:47,560 But if it has the plasma, it encodes this gene 81 00:05:47,560 --> 00:05:50,380 that confers ampicillin resistance, 82 00:05:50,380 --> 00:05:53,530 and it will thus be able to grow. 83 00:05:53,530 --> 00:05:54,860 So that's exactly right. 84 00:05:54,860 --> 00:05:59,950 So you're able to select for bacteria with a given plasmid 85 00:05:59,950 --> 00:06:06,280 by simply growing it on media that contains an antibiotic. 86 00:06:06,280 --> 00:06:11,410 I now want to go through steps in cloning a piece of DNA. 87 00:06:11,410 --> 00:06:15,960 And we'll go through it sort of as a series of ordered steps 88 00:06:15,960 --> 00:06:18,980 so you can see how the process works. 89 00:06:18,980 --> 00:06:23,155 I'm going to start with a step to cut the DNA. 90 00:06:25,930 --> 00:06:29,585 After cutting the DNA, we'll then mix pieces together. 91 00:06:32,440 --> 00:06:34,930 Once we mix the pieces together, we'll 92 00:06:34,930 --> 00:06:37,060 do something known as a ligation, 93 00:06:37,060 --> 00:06:41,030 and I'll explain that to you in just a minute. 94 00:06:41,030 --> 00:06:44,470 And then, finally, we'll end with selecting 95 00:06:44,470 --> 00:06:51,140 the plasmids that have the piece of foreign DNA that we want. 96 00:06:51,140 --> 00:06:53,650 And this is known as recombinant DNA, 97 00:06:53,650 --> 00:06:57,130 because you've recombined a piece of DNA from one 98 00:06:57,130 --> 00:06:59,890 organism-- it could be a eukaryotic organism, 99 00:06:59,890 --> 00:07:01,630 like humans-- 100 00:07:01,630 --> 00:07:05,110 with a piece of DNA that's from a prokaryotic cell, 101 00:07:05,110 --> 00:07:07,360 a bacterium. 102 00:07:07,360 --> 00:07:09,940 So we then have some sort of selection process. 103 00:07:13,540 --> 00:07:17,260 So we're going to go through this step-by-step. 104 00:07:17,260 --> 00:07:20,180 And we're going to start with cutting DNA, OK? 105 00:07:23,590 --> 00:07:24,535 So, cutting DNA. 106 00:07:30,040 --> 00:07:33,070 And it turns out, we've talked about-- 107 00:07:33,070 --> 00:07:36,130 what type of enzyme do you think would cut DNA? 108 00:07:42,200 --> 00:07:45,590 Just generally, not as specific as what's up on the slide. 109 00:07:45,590 --> 00:07:49,520 What type of enzyme would cleave DNA? 110 00:07:49,520 --> 00:07:54,282 Think about how enzymes are named. 111 00:07:54,282 --> 00:07:55,240 AUDIENCE: DNase? 112 00:07:55,240 --> 00:07:57,020 ADAM MARTIN: Yeah, so Stephen suggested 113 00:07:57,020 --> 00:08:00,590 DNase, which is a really good suggestion, right? 114 00:08:00,590 --> 00:08:05,090 So an enzyme that will cut DNA would be a DNase. 115 00:08:05,090 --> 00:08:07,010 Another word for that is a nuclease. 116 00:08:07,010 --> 00:08:08,950 It's some type of nuclease. 117 00:08:08,950 --> 00:08:12,140 And the type of nuclease we're going to talk about 118 00:08:12,140 --> 00:08:16,040 is going to be an endonuclease. 119 00:08:16,040 --> 00:08:18,200 We talked about exonucleases, which 120 00:08:18,200 --> 00:08:23,640 chew DNA from the ends in the context of DNA replication. 121 00:08:23,640 --> 00:08:26,330 But what an endonuclease is, is it's 122 00:08:26,330 --> 00:08:34,590 a nuclease that's going to recognize a fragment of DNA 123 00:08:34,590 --> 00:08:36,000 and cleave it in the middle. 124 00:08:36,000 --> 00:08:37,740 So it doesn't require an end. 125 00:08:37,740 --> 00:08:41,250 It's going to chop it right in the middle. 126 00:08:41,250 --> 00:08:45,370 And there's a type of endonuclease, 127 00:08:45,370 --> 00:08:48,630 and these are called restriction endonucleases. 128 00:08:48,630 --> 00:08:51,300 They are nucleases that our natively present 129 00:08:51,300 --> 00:08:55,070 in a lot of different bacteria. 130 00:08:55,070 --> 00:08:58,370 And these restriction endonucleases essentially 131 00:08:58,370 --> 00:09:01,010 look through the DNA sequence, and they 132 00:09:01,010 --> 00:09:06,290 recognize a specific sequence of nucleotides 133 00:09:06,290 --> 00:09:10,070 and make a cut right at that sequence. 134 00:09:10,070 --> 00:09:12,870 So I have a few examples to show you here. 135 00:09:12,870 --> 00:09:17,180 The first is this EcoR1 restriction endonuclease. 136 00:09:17,180 --> 00:09:24,760 EcoR1's from E. coli, and it recognizes the sequence GAATTC. 137 00:09:24,760 --> 00:09:27,320 So it recognizes this six-nucleotide . 138 00:09:27,320 --> 00:09:31,160 Sequence and it cleaves the double-stranded DNA 139 00:09:31,160 --> 00:09:33,985 on the top strand between the G and the A 140 00:09:33,985 --> 00:09:37,880 and on the bottom strand between the G and the A, OK? 141 00:09:37,880 --> 00:09:40,790 And you can see that the two cuts are staggered. 142 00:09:40,790 --> 00:09:45,770 So when this cut is made, it leaves the DNA with two ends, 143 00:09:45,770 --> 00:09:51,020 and they're sticky, because there's a five-prime overhang 144 00:09:51,020 --> 00:09:52,430 at each end. 145 00:09:52,430 --> 00:09:55,805 So each end has this TTAA sequence. 146 00:09:58,850 --> 00:10:01,700 And these nucleotide bases can base pair 147 00:10:01,700 --> 00:10:04,910 with the complementary sequence. 148 00:10:04,910 --> 00:10:08,030 So this sequence could base pair with a sequence 149 00:10:08,030 --> 00:10:09,290 that has an end AATT. 150 00:10:11,900 --> 00:10:15,290 So these two ends that I've generated here 151 00:10:15,290 --> 00:10:17,210 could stick to each other, or there 152 00:10:17,210 --> 00:10:21,560 could be other ends that have the TTAA sequence that 153 00:10:21,560 --> 00:10:24,590 could stick to them. 154 00:10:24,590 --> 00:10:28,880 So another example is this Kpn1 endonuclease. 155 00:10:28,880 --> 00:10:31,280 And this is from a different bacterium. 156 00:10:31,280 --> 00:10:35,000 But again, it cleaves the DNA on the two strands. 157 00:10:35,000 --> 00:10:38,270 And this time, it cleaves the top strand 158 00:10:38,270 --> 00:10:40,370 farther down the sequence. 159 00:10:40,370 --> 00:10:43,440 And that generates what's known as a three-prime overhang. 160 00:10:43,440 --> 00:10:45,260 But again, you have an overhang. 161 00:10:45,260 --> 00:10:48,410 So this is what is known as having a sticky end. 162 00:10:48,410 --> 00:10:50,840 Because again, these nucleotides are 163 00:10:50,840 --> 00:10:55,430 available to base pair with a complementary sequence. 164 00:10:55,430 --> 00:10:57,560 The final type of restriction enzyme 165 00:10:57,560 --> 00:11:00,560 that I'll tell you about is EcoR5, 166 00:11:00,560 --> 00:11:03,770 which is a different enzyme from E. coli. 167 00:11:03,770 --> 00:11:06,650 And this generates a break, but this time, it 168 00:11:06,650 --> 00:11:10,700 cuts at the same position on both DNA strands. 169 00:11:10,700 --> 00:11:14,390 And that generates an end that's known as a blunt end, 170 00:11:14,390 --> 00:11:16,010 because there's no overhang, and there 171 00:11:16,010 --> 00:11:20,270 are no nucleotides that would sort of basically 172 00:11:20,270 --> 00:11:25,890 recognize a complementary sort of end like the sticky ends do, 173 00:11:25,890 --> 00:11:27,620 OK? 174 00:11:27,620 --> 00:11:31,130 So these are several of many, many different types 175 00:11:31,130 --> 00:11:33,680 of restriction endonucleases that 176 00:11:33,680 --> 00:11:35,825 are present in a wide range of bacteria. 177 00:11:39,830 --> 00:11:42,770 So then, now that you have a tool that 178 00:11:42,770 --> 00:11:46,130 allows you to cut DNA, you could then cut DNA 179 00:11:46,130 --> 00:11:47,810 from two different sources. 180 00:11:47,810 --> 00:11:50,300 And I've outlined that here. 181 00:11:50,300 --> 00:11:56,060 The vector is what the plasmid DNA is commonly referred to. 182 00:11:56,060 --> 00:11:59,810 So we commonly refer to this prokaryotic part 183 00:11:59,810 --> 00:12:03,500 of the plasmid, the vector DNA, and the part 184 00:12:03,500 --> 00:12:07,060 that we're trying to insert that's the foreign DNA, 185 00:12:07,060 --> 00:12:09,920 the insert. 186 00:12:09,920 --> 00:12:11,795 That's just kind of the lingo in the field. 187 00:12:14,600 --> 00:12:17,300 So here, I have a plasmid. 188 00:12:17,300 --> 00:12:18,440 It looks like a plasmid. 189 00:12:18,440 --> 00:12:20,190 It has an origin of replication. 190 00:12:20,190 --> 00:12:22,400 It has ampicillin resistance. 191 00:12:22,400 --> 00:12:26,270 And it has this EcoR1 site, which just means that this DNA 192 00:12:26,270 --> 00:12:30,380 sequence has a GAATTC, OK? 193 00:12:30,380 --> 00:12:33,260 So it's something that will be recognized by this restriction 194 00:12:33,260 --> 00:12:34,940 endonuclease. 195 00:12:34,940 --> 00:12:39,200 And then if you cut this enzyme with EcoR1, 196 00:12:39,200 --> 00:12:41,360 you start with the linear piece, right? 197 00:12:41,360 --> 00:12:47,060 So I, at 6:00 AM, started engineering this here. 198 00:12:47,060 --> 00:12:50,570 So let's say I have my plasmid DNA, 199 00:12:50,570 --> 00:12:52,700 and I cut it at the EcoR1 site. 200 00:12:56,220 --> 00:12:57,500 Then I cleave it. 201 00:12:57,500 --> 00:13:01,770 If you cleave a circle, now you have a linear piece of DNA, OK? 202 00:13:01,770 --> 00:13:04,620 But it has sticky ends, right? 203 00:13:04,620 --> 00:13:07,260 And these sticky ends-- 204 00:13:07,260 --> 00:13:11,110 so pretend that this is a foreign piece of DNA. 205 00:13:11,110 --> 00:13:13,440 This is my eukaryotic DNA. 206 00:13:13,440 --> 00:13:16,890 Let's pretend it carries the gene elastin. 207 00:13:16,890 --> 00:13:19,410 And this has ends, too. 208 00:13:19,410 --> 00:13:23,310 And if they're EcoR1 ends, then they 209 00:13:23,310 --> 00:13:27,540 will be able to stick to the sticky ends of the plasmid. 210 00:13:27,540 --> 00:13:29,370 And if you just get one sticking, 211 00:13:29,370 --> 00:13:32,100 now you have this piece of DNA which 212 00:13:32,100 --> 00:13:34,950 is two different fragments in tandem. 213 00:13:34,950 --> 00:13:36,720 But it's going to be moving around 214 00:13:36,720 --> 00:13:38,850 in space in the cytoplasm. 215 00:13:38,850 --> 00:13:43,140 And at some point, it might be recognized and stick 216 00:13:43,140 --> 00:13:45,450 to the other EcoR1 end. 217 00:13:45,450 --> 00:13:49,860 And then you have now a circular piece of DNA again, 218 00:13:49,860 --> 00:13:52,440 but now your circular piece of DNA 219 00:13:52,440 --> 00:13:55,080 has this piece of foreign DNA that's 220 00:13:55,080 --> 00:13:59,100 present inside the vector, which is the sort of poster tape 221 00:13:59,100 --> 00:14:00,630 here. 222 00:14:00,630 --> 00:14:02,240 So I just wanted to show you that. 223 00:14:02,240 --> 00:14:03,690 So you can kind of-- 224 00:14:03,690 --> 00:14:05,190 when you're doing molecular biology, 225 00:14:05,190 --> 00:14:06,960 you have to imagine the sort of end 226 00:14:06,960 --> 00:14:09,270 stick sticking to each other and how they're 227 00:14:09,270 --> 00:14:12,870 going to sort of wrap around and connect for the final product. 228 00:14:16,590 --> 00:14:20,070 OK, so let's say you get DNA, and your DNA could 229 00:14:20,070 --> 00:14:25,290 be eukaryotic DNA from, let's say, humans or flies 230 00:14:25,290 --> 00:14:28,680 or whatever your favorite eukaryote is. 231 00:14:28,680 --> 00:14:32,700 And in the genome of that organism, 232 00:14:32,700 --> 00:14:35,640 there will be many restriction sites. 233 00:14:35,640 --> 00:14:38,010 But if you chop it up, you will get 234 00:14:38,010 --> 00:14:42,420 various fragments that have sticky ends for EcoR1 235 00:14:42,420 --> 00:14:43,890 on both sides. 236 00:14:43,890 --> 00:14:47,730 And then if you mixed the vector and the insert together, 237 00:14:47,730 --> 00:14:51,030 you have some probability of getting that insert 238 00:14:51,030 --> 00:14:55,860 to be incorporated into the vector. 239 00:14:55,860 --> 00:14:59,220 And then once this is present and ligated together, 240 00:14:59,220 --> 00:15:01,140 you can then put this into bacteria, 241 00:15:01,140 --> 00:15:02,490 and it will be replicated. 242 00:15:11,910 --> 00:15:16,770 So we focused on cutting, but if you mix these together, 243 00:15:16,770 --> 00:15:18,990 like I showed you at the tape, you're 244 00:15:18,990 --> 00:15:21,210 going to have sticky ends that come together 245 00:15:21,210 --> 00:15:23,040 and stick together. 246 00:15:23,040 --> 00:15:25,623 And you'll eventually get a situation 247 00:15:25,623 --> 00:15:26,790 where you have your insert-- 248 00:15:30,900 --> 00:15:32,910 so you have your insert here that 249 00:15:32,910 --> 00:15:37,080 might have your gene of interest and your vector DNA here. 250 00:15:37,080 --> 00:15:39,960 But when these things initially stick together, 251 00:15:39,960 --> 00:15:44,130 you don't have a single molecule where everything 252 00:15:44,130 --> 00:15:45,780 is covalently attached, right? 253 00:15:45,780 --> 00:15:49,890 You just have these base pair interactions between the two 254 00:15:49,890 --> 00:15:52,470 overhangs as they stick to each other 255 00:15:52,470 --> 00:15:55,540 through base pair interactions. 256 00:15:55,540 --> 00:15:58,530 So if we think about what's going on right here, 257 00:15:58,530 --> 00:16:03,560 you have, initially, if we're thinking about EcoR1, 258 00:16:03,560 --> 00:16:05,900 a sequence that is-- 259 00:16:05,900 --> 00:16:09,900 oh, sorry, this should be a C. This 260 00:16:09,900 --> 00:16:13,680 is the nucleotide sequence, but when it's sticking together, 261 00:16:13,680 --> 00:16:18,630 the top strand will have a free five-prime phosphate 262 00:16:18,630 --> 00:16:20,400 on this adenosine, and there will 263 00:16:20,400 --> 00:16:25,470 be no covalent bond between this adenosine and this guanosine. 264 00:16:25,470 --> 00:16:27,810 So that's what the top strand would look like. 265 00:16:27,810 --> 00:16:30,260 The bottom strand would look like this, 266 00:16:30,260 --> 00:16:35,640 where there are covalent bonds along the DNA backbone. 267 00:16:35,640 --> 00:16:37,830 Sorry. 268 00:16:37,830 --> 00:16:41,670 Had a mutation there. 269 00:16:41,670 --> 00:16:45,880 And this is incorporated in a broader sequence. 270 00:16:45,880 --> 00:16:49,110 And this bottom strand will have a free five-prime phosphate 271 00:16:49,110 --> 00:16:53,160 here and a free three-prime hydroxyl here, 272 00:16:53,160 --> 00:16:55,300 but there's no covalent bond here. 273 00:16:55,300 --> 00:16:57,240 There's no covalent bond here, right? 274 00:16:57,240 --> 00:17:01,240 So, at this stage, you don't have a single piece of DNA. 275 00:17:01,240 --> 00:17:05,130 You have two pieces of DNA that are interacting with each other 276 00:17:05,130 --> 00:17:07,650 through base pair interactions. 277 00:17:07,650 --> 00:17:11,290 So, eventually, you want it to be a single piece of DNA. 278 00:17:11,290 --> 00:17:14,940 And so you have to perform a step that is known as-- 279 00:17:14,940 --> 00:17:18,460 sorry, my phosphate got in the way. 280 00:17:18,460 --> 00:17:21,750 But you want to perform what's known as a ligation, where you 281 00:17:21,750 --> 00:17:24,750 take something that's just sticking together 282 00:17:24,750 --> 00:17:28,020 through nucleotide base pair interactions 283 00:17:28,020 --> 00:17:31,050 and you add a type of enzyme, which 284 00:17:31,050 --> 00:17:34,560 is called DNA ligase, to catalyze 285 00:17:34,560 --> 00:17:39,630 the formation of covalent bonds here and here. 286 00:17:39,630 --> 00:17:43,380 So DNA ligase is an enzyme that if you 287 00:17:43,380 --> 00:17:45,810 have a three five-prime phosphate 288 00:17:45,810 --> 00:17:48,300 here and a free three-prime hydroxyl, 289 00:17:48,300 --> 00:17:51,480 it'll catalyze the formation of a phosphodiester 290 00:17:51,480 --> 00:17:56,460 bond between these two bases and the DNA. 291 00:17:56,460 --> 00:18:00,525 So this DNA ligase forms a phosphodiester bond. 292 00:18:08,310 --> 00:18:13,110 And you go from having no bond there to having a bond. 293 00:18:13,110 --> 00:18:19,140 So then you eventually would get this sequence now, 294 00:18:19,140 --> 00:18:26,640 where you have covalent bonds between each of the base pairs. 295 00:18:26,640 --> 00:18:31,070 And what you see here is you've recreated the EcoR1 site. 296 00:18:31,070 --> 00:18:35,040 So when you get two EcoR1 sticky ends 297 00:18:35,040 --> 00:18:38,220 sort of recognizing each other and sticking to each other 298 00:18:38,220 --> 00:18:40,290 and then you ligate them together, 299 00:18:40,290 --> 00:18:44,700 you recreate that nuclease site so that you can cleave it again 300 00:18:44,700 --> 00:18:46,200 with the EcoR1 enzyme. 301 00:18:51,770 --> 00:18:53,450 So now, moving on. 302 00:18:53,450 --> 00:18:55,680 I'll move on right here. 303 00:18:55,680 --> 00:19:01,490 So the last step is that once we have pieces of DNA 304 00:19:01,490 --> 00:19:02,270 with this insert-- 305 00:19:04,940 --> 00:19:09,270 and let's say we're trying to find a piece of DNA 306 00:19:09,270 --> 00:19:11,640 from a eukaryotic organism. 307 00:19:11,640 --> 00:19:13,680 We might start with an animal. 308 00:19:13,680 --> 00:19:16,590 We have to extract its chromosomal DNA, 309 00:19:16,590 --> 00:19:19,260 digest it with a restriction enzyme, 310 00:19:19,260 --> 00:19:22,200 and then we digest the vector with the same restriction 311 00:19:22,200 --> 00:19:23,190 enzyme. 312 00:19:23,190 --> 00:19:24,920 And then we're going to make-- 313 00:19:24,920 --> 00:19:27,810 we're going to randomly insert these pieces of DNA 314 00:19:27,810 --> 00:19:31,320 into different vectors such that each bacteria who 315 00:19:31,320 --> 00:19:33,990 gets one of these plasmids will be 316 00:19:33,990 --> 00:19:36,810 replicating a different piece of DNA that's 317 00:19:36,810 --> 00:19:39,840 of eukaryotic origin. 318 00:19:39,840 --> 00:19:42,060 And this is what is known as a DNA library. 319 00:19:44,680 --> 00:19:46,440 So this is making a DNA library. 320 00:19:49,680 --> 00:19:52,410 And a DNA library is essentially a collection 321 00:19:52,410 --> 00:19:57,840 of different pieces of DNA that are from some source, OK? 322 00:19:57,840 --> 00:20:01,110 But different bacterial clones will be replicating 323 00:20:01,110 --> 00:20:05,220 a different piece of that DNA. 324 00:20:05,220 --> 00:20:08,010 So you can see the challenge now is 325 00:20:08,010 --> 00:20:11,460 to find the needle in the haystack, right? 326 00:20:11,460 --> 00:20:14,400 You're trying to find that one piece of DNA which is 327 00:20:14,400 --> 00:20:17,550 the one you're interested in. 328 00:20:17,550 --> 00:20:20,580 And I'll talk about several strategies 329 00:20:20,580 --> 00:20:23,730 that you can use to find the piece of DNA 330 00:20:23,730 --> 00:20:25,770 that you're interested in. 331 00:20:25,770 --> 00:20:27,210 I'll focus on selection. 332 00:20:27,210 --> 00:20:29,100 But first, I just want to differentiate 333 00:20:29,100 --> 00:20:31,500 between two different types of ways you could 334 00:20:31,500 --> 00:20:33,940 search for a piece of DNA. 335 00:20:33,940 --> 00:20:35,460 You could do a screen. 336 00:20:35,460 --> 00:20:38,760 And this is similar to what we talked about on Monday, where 337 00:20:38,760 --> 00:20:42,420 you look through a whole population of individuals, 338 00:20:42,420 --> 00:20:44,610 and you look for a given phenotype. 339 00:20:44,610 --> 00:20:48,360 So in the case of flies, we talked 340 00:20:48,360 --> 00:20:50,640 about how Morgan's lab was looking 341 00:20:50,640 --> 00:20:53,190 for differences in eye color. 342 00:20:53,190 --> 00:20:54,870 And that was a screen, because they 343 00:20:54,870 --> 00:20:56,820 looked through a ton of normal flies 344 00:20:56,820 --> 00:20:58,860 to find the one they want. 345 00:20:58,860 --> 00:21:01,290 Another strategy would be to do what's 346 00:21:01,290 --> 00:21:06,810 called a selection, where you kill off everything that's not 347 00:21:06,810 --> 00:21:10,770 what you want by making the organism grow 348 00:21:10,770 --> 00:21:14,220 in some sort of selective condition. 349 00:21:14,220 --> 00:21:16,320 And then you only allow the organisms 350 00:21:16,320 --> 00:21:18,810 to grow that are the ones that you want. 351 00:21:18,810 --> 00:21:21,860 So this is called a selection. 352 00:21:21,860 --> 00:21:25,200 And I'm going to illustrate several examples of selections, 353 00:21:25,200 --> 00:21:28,920 just so that you get an idea of how this works. 354 00:21:28,920 --> 00:21:34,290 So the first example I'll give is antibiotic resistance. 355 00:21:40,140 --> 00:21:43,620 And as Stephen so kindly pointed out earlier in the lecture, 356 00:21:43,620 --> 00:21:46,950 the way we can select for bacteria that have taken up 357 00:21:46,950 --> 00:21:50,220 this plasmid is to select the bacteria that 358 00:21:50,220 --> 00:21:56,580 grow in the presence of the antibiotic. 359 00:21:56,580 --> 00:21:59,580 So let's say you had a population of bacteria 360 00:21:59,580 --> 00:22:01,200 and let's say this started out being 361 00:22:01,200 --> 00:22:04,080 sensitive to an antibiotic. 362 00:22:04,080 --> 00:22:07,680 You could transform them with DNA 363 00:22:07,680 --> 00:22:09,570 from a strain that's resistant. 364 00:22:14,670 --> 00:22:18,480 And maybe that resistant strain has a plasmid 365 00:22:18,480 --> 00:22:23,160 that has a gene that confers antibiotic resistance, in which 366 00:22:23,160 --> 00:22:27,030 case, if it's taken up by this sensitive bacteria, 367 00:22:27,030 --> 00:22:34,410 if you then grow it on a plate that has the antibiotic, 368 00:22:34,410 --> 00:22:37,170 you might get a colony or a clone of cells 369 00:22:37,170 --> 00:22:39,210 that has taken up the piece of DNA 370 00:22:39,210 --> 00:22:43,050 that you're interested in-- in this case, the piece of DNA 371 00:22:43,050 --> 00:22:47,070 that confers antibiotic resistance. 372 00:22:47,070 --> 00:22:49,180 Everyone see how that works? 373 00:22:49,180 --> 00:22:51,390 So you're selecting only the cells 374 00:22:51,390 --> 00:22:54,910 to grow here that have taken up this antibiotic resistance 375 00:22:54,910 --> 00:22:55,410 gene. 376 00:22:58,890 --> 00:23:02,430 I'm going to use another example now from yeast, 377 00:23:02,430 --> 00:23:04,840 and it involves functional complementation. 378 00:23:11,380 --> 00:23:14,490 And I'm going to start with something 379 00:23:14,490 --> 00:23:18,240 that involves the biosynthesis of an essential amino acid. 380 00:23:18,240 --> 00:23:21,360 And then I'm going to go to a more interesting case, which 381 00:23:21,360 --> 00:23:25,050 is a case that involves an experiment that involved 382 00:23:25,050 --> 00:23:28,410 the identification of the master regulator of cell division 383 00:23:28,410 --> 00:23:29,790 in humans. 384 00:23:29,790 --> 00:23:33,890 But we'll start with just amino acid biosynthesis. 385 00:23:33,890 --> 00:23:39,090 And there are mutants of yeast known as auxotrophs. 386 00:23:39,090 --> 00:23:44,820 And these are mutant yeast, or mutant microorganisms, 387 00:23:44,820 --> 00:23:48,360 that fail to produce an essential nutrient. 388 00:23:48,360 --> 00:23:53,820 So an auxotroph is a mutant that fails to make 389 00:23:53,820 --> 00:23:59,050 a nutrient that's essential. 390 00:23:59,050 --> 00:24:02,940 So fails to make a nutrient. 391 00:24:02,940 --> 00:24:09,630 And the opposite of an auxotroph is called a prototroph. 392 00:24:09,630 --> 00:24:18,150 A prototroph is essentially a normal-functioning 393 00:24:18,150 --> 00:24:20,760 microorganism that's able to produce 394 00:24:20,760 --> 00:24:24,270 all of the essential nutrients that it needs in order 395 00:24:24,270 --> 00:24:26,550 to grow and survive, OK? 396 00:24:26,550 --> 00:24:29,160 So this produces all nutrients. 397 00:24:36,830 --> 00:24:41,340 And so let's say you had an auxotroph for leucine. 398 00:24:41,340 --> 00:24:45,990 So you had a strain that if you didn't provide leucine to it, 399 00:24:45,990 --> 00:24:48,690 it would fail to grow. 400 00:24:48,690 --> 00:24:52,080 So we'll start with a leucine auxotroph. 401 00:24:54,840 --> 00:24:57,120 And let's say you want to identify 402 00:24:57,120 --> 00:25:01,170 the gene that's defective in this leucine auxotroph. 403 00:25:01,170 --> 00:25:06,000 You'd perform a similar strategy to this one, where 404 00:25:06,000 --> 00:25:10,620 you'd have your auxotroph that you're transforming, 405 00:25:10,620 --> 00:25:13,260 so your auxotroph down here. 406 00:25:13,260 --> 00:25:16,740 And you would transform that strain with DNA 407 00:25:16,740 --> 00:25:18,270 from what organism? 408 00:25:21,530 --> 00:25:24,590 If you're trying to identify the functional gene, 409 00:25:24,590 --> 00:25:27,230 what organism would you use to produce 410 00:25:27,230 --> 00:25:30,654 the DNA you're going to transform into that organism? 411 00:25:30,654 --> 00:25:31,590 AUDIENCE: Prototroph? 412 00:25:31,590 --> 00:25:33,440 ADAM MARTIN: Javier's exactly right. 413 00:25:33,440 --> 00:25:35,390 You'd use the prototroph, right? 414 00:25:35,390 --> 00:25:40,470 So in this case, you would use DNA from the prototroph, 415 00:25:40,470 --> 00:25:44,120 because the prototroph has a functional copy of that gene. 416 00:25:44,120 --> 00:25:46,220 You know it does, because it's able to grow 417 00:25:46,220 --> 00:25:48,680 without adding leucine. 418 00:25:48,680 --> 00:25:54,290 And then you could take the auxotroph mutants that's 419 00:25:54,290 --> 00:25:57,260 transformed with DNA from the prototroph, 420 00:25:57,260 --> 00:26:00,560 and you played it on media. 421 00:26:00,560 --> 00:26:04,460 And what should be a property of the media? 422 00:26:04,460 --> 00:26:06,700 Should leucine be present or absent? 423 00:26:06,700 --> 00:26:07,272 Carmen? 424 00:26:07,272 --> 00:26:07,980 AUDIENCE: Absent. 425 00:26:07,980 --> 00:26:11,120 ADAM MARTIN: It should be absent, exactly right. 426 00:26:11,120 --> 00:26:13,370 So you'd look on plates that lack 427 00:26:13,370 --> 00:26:16,010 leucine or minus for leucine. 428 00:26:16,010 --> 00:26:19,160 And you'd select for colonies that now are all of a sudden 429 00:26:19,160 --> 00:26:22,040 able to grow leucine. 430 00:26:22,040 --> 00:26:27,220 So you've restored the function of leucine biosynthesis 431 00:26:27,220 --> 00:26:30,860 in this clone, and you've made it into a prototroph again. 432 00:26:34,870 --> 00:26:38,540 OK, this is what's known as functional complementation, 433 00:26:38,540 --> 00:26:40,580 because you're taking a cell that 434 00:26:40,580 --> 00:26:45,610 is defective in some function, and you're complementing it. 435 00:26:45,610 --> 00:26:51,470 You're complementing or rescuing the phenotype, OK? 436 00:26:51,470 --> 00:26:54,740 Now, even as a former yeast geneticist, 437 00:26:54,740 --> 00:26:59,060 I don't find amino acid biosynthesis and functional 438 00:26:59,060 --> 00:27:03,300 complementation in the context of leucine all that exciting. 439 00:27:03,300 --> 00:27:06,650 So I want to present one last example that 440 00:27:06,650 --> 00:27:13,220 involves an experiment that is going to involve the yeast cell 441 00:27:13,220 --> 00:27:13,940 cycle mutants. 442 00:27:17,360 --> 00:27:20,720 And I'm going to tell you about the experiment that 443 00:27:20,720 --> 00:27:26,210 led to the cloning of the master regulator of cell division 444 00:27:26,210 --> 00:27:27,830 in humans. 445 00:27:27,830 --> 00:27:33,080 And it involves a yeast mutant, and specifically, a yeast cell 446 00:27:33,080 --> 00:27:35,390 cycle mutant. 447 00:27:35,390 --> 00:27:37,940 And these yeast cell cycle mutants 448 00:27:37,940 --> 00:27:41,420 are what are known as conditional mutants. 449 00:27:41,420 --> 00:27:44,810 They are isolated as conditional mutants, 450 00:27:44,810 --> 00:27:47,510 meaning that these mutants are able to grow 451 00:27:47,510 --> 00:27:50,660 under certain conditions, but not others. 452 00:27:50,660 --> 00:27:55,040 And specifically, the condition they used is temperature, 453 00:27:55,040 --> 00:27:57,470 so they're temperature-sensitive mutants. 454 00:27:57,470 --> 00:28:06,020 The yeast cells can grow at 25 degrees, but not at 37 degrees. 455 00:28:06,020 --> 00:28:09,110 So this is known as a temperature-sensitive mutant, 456 00:28:09,110 --> 00:28:12,200 where you can propagate the mutant at one temperature, 457 00:28:12,200 --> 00:28:14,540 but then you can see if you raise the temperature, 458 00:28:14,540 --> 00:28:16,730 then it stops growing. 459 00:28:16,730 --> 00:28:18,530 And so you can see the mutant phenotype, 460 00:28:18,530 --> 00:28:21,530 because normal wild type functional 461 00:28:21,530 --> 00:28:24,170 yeast can grow at both temperatures. 462 00:28:24,170 --> 00:28:28,190 So this is a special type of mutant. 463 00:28:28,190 --> 00:28:30,440 And I'm going to tell you about an experiment done 464 00:28:30,440 --> 00:28:36,890 by Paul Nurse, who's an excellent yeast geneticist. 465 00:28:36,890 --> 00:28:41,840 And what he did was he used these yeast cell cycle mutants 466 00:28:41,840 --> 00:28:45,860 to identify the human gene for what's 467 00:28:45,860 --> 00:28:57,860 now known as cyclin-dependent kinase, or CDK for short. 468 00:28:57,860 --> 00:29:01,010 This is the master regulator of cell division 469 00:29:01,010 --> 00:29:05,810 in organisms ranging from yeast all the way up to humans, OK? 470 00:29:05,810 --> 00:29:11,210 But he used yeast as a model system to identify this gene. 471 00:29:11,210 --> 00:29:14,600 And the process was he took yeast cells-- 472 00:29:14,600 --> 00:29:16,820 and Paul Nurse worked on fission yeast cells, 473 00:29:16,820 --> 00:29:18,980 which are rod-shaped cells. 474 00:29:18,980 --> 00:29:20,855 And he identified yeast mutants. 475 00:29:24,590 --> 00:29:25,370 Yeast mutants. 476 00:29:25,370 --> 00:29:30,530 And he had a mutant in the CDK gene of yeast. 477 00:29:30,530 --> 00:29:32,700 He didn't really know it at the time. 478 00:29:32,700 --> 00:29:36,710 But the yeast CDK mutant-- 479 00:29:36,710 --> 00:29:40,010 what he knew was that this mutant was critically involved 480 00:29:40,010 --> 00:29:42,770 in the cell cycle in numerous types of yeast. 481 00:29:42,770 --> 00:29:44,960 So he knew this is an important gene. 482 00:29:44,960 --> 00:29:47,780 And what he wanted to do was to identify 483 00:29:47,780 --> 00:29:50,030 if humans had an equivalent gene that 484 00:29:50,030 --> 00:29:53,780 could function in the same way. 485 00:29:53,780 --> 00:29:57,530 So if you just have this CDK mutant and do nothing to it, 486 00:29:57,530 --> 00:30:01,160 it will not grow at 37 degrees, OK? 487 00:30:01,160 --> 00:30:05,990 But what he did was to take a DNA library-- 488 00:30:05,990 --> 00:30:07,670 similar to what I showed you before, 489 00:30:07,670 --> 00:30:11,900 where you just chop up DNA from an organism. 490 00:30:11,900 --> 00:30:15,090 In this case, he's using a human DNA library. 491 00:30:15,090 --> 00:30:17,060 And he used a particular type of library, 492 00:30:17,060 --> 00:30:19,135 but I'm going to skip over that for now 493 00:30:19,135 --> 00:30:21,040 and come back to it later. 494 00:30:21,040 --> 00:30:25,170 So he used a human DNA library. 495 00:30:25,170 --> 00:30:26,910 That's just a collection of pieces 496 00:30:26,910 --> 00:30:30,210 of DNA from a human source, OK? 497 00:30:30,210 --> 00:30:33,780 So he's taking human DNA, putting it 498 00:30:33,780 --> 00:30:36,930 into a yeast plasmid, and transforming yeast 499 00:30:36,930 --> 00:30:38,790 with that human DNA. 500 00:30:38,790 --> 00:30:41,370 And he's looking for a piece of DNA 501 00:30:41,370 --> 00:30:45,330 that's able to complement the CDK mutant, 502 00:30:45,330 --> 00:30:47,220 meaning the yeast cells would then 503 00:30:47,220 --> 00:30:50,250 be able to grow at the non-permissive temperature 504 00:30:50,250 --> 00:30:53,070 of 37 degrees. 505 00:30:53,070 --> 00:30:57,390 So he then looks for, on a plate, colonies of yeast 506 00:30:57,390 --> 00:31:00,030 that are growing at the non-permissive temperature 507 00:31:00,030 --> 00:31:01,810 of 37 degrees. 508 00:31:01,810 --> 00:31:03,930 So if you didn't do anything with this mutant, 509 00:31:03,930 --> 00:31:07,020 if you didn't transform in the DNA, nothing would grow. 510 00:31:07,020 --> 00:31:10,230 But he identified pieces of human DNA 511 00:31:10,230 --> 00:31:16,310 that rescued the phenotype of this mutant, OK? 512 00:31:16,310 --> 00:31:24,930 And so these are yeast that have the human gene for CDK, 513 00:31:24,930 --> 00:31:27,690 and they now grow. 514 00:31:27,690 --> 00:31:31,050 And this is a functional complementation experiment, 515 00:31:31,050 --> 00:31:34,750 because you're rescuing the growth of this yeast 516 00:31:34,750 --> 00:31:38,850 now not with a yeast gene, but with a human gene. 517 00:31:38,850 --> 00:31:43,770 And this human CDK gene is so conserved across eukaryotes 518 00:31:43,770 --> 00:31:47,010 that it's able to still function in a yeast cell, which 519 00:31:47,010 --> 00:31:48,940 is pretty amazing. 520 00:31:48,940 --> 00:31:51,996 So this just outlines the experiment here. 521 00:31:51,996 --> 00:31:54,690 At 25 degrees, these yeast mutants 522 00:31:54,690 --> 00:31:57,750 can grow and form colonies. 523 00:31:57,750 --> 00:32:00,900 And at that temperature, you can transform the yeast 524 00:32:00,900 --> 00:32:04,650 with different pieces of human DNA. 525 00:32:04,650 --> 00:32:07,530 Most of the human DNA is not going to be what you want. 526 00:32:07,530 --> 00:32:09,660 You're looking for the needle in the haystack. 527 00:32:09,660 --> 00:32:12,810 So most of these are not going to grow at 37 degree. 528 00:32:12,810 --> 00:32:16,950 But you're looking for this guy here that gets the human CDK, 529 00:32:16,950 --> 00:32:21,750 and that restores growth to this mutant strain. 530 00:32:21,750 --> 00:32:25,320 So voila, you get a colony of cells that are growing. 531 00:32:25,320 --> 00:32:28,020 And boom, Paul Nurse wins a Nobel Prize 532 00:32:28,020 --> 00:32:31,440 and the rest of the yeast field, as well, or a number of people 533 00:32:31,440 --> 00:32:33,990 who are working on cell cycle mutants. 534 00:32:33,990 --> 00:32:35,820 This is one of the experiments that 535 00:32:35,820 --> 00:32:41,370 led to the 2001 Nobel Prize for a bunch of yeast cell cycle 536 00:32:41,370 --> 00:32:44,360 geneticists. 537 00:32:44,360 --> 00:32:46,710 All right, so I've told you about how to find 538 00:32:46,710 --> 00:32:47,835 the needle in the haystack. 539 00:32:50,730 --> 00:32:55,500 And this was more common when we didn't know the genome sequence 540 00:32:55,500 --> 00:32:56,760 of an organism. 541 00:32:56,760 --> 00:33:00,420 But now I want to tell you how knowing the genome sequence 542 00:33:00,420 --> 00:33:04,620 of an organism would allow you to replicate and amplify 543 00:33:04,620 --> 00:33:11,280 a piece of DNA in vitro. 544 00:33:11,280 --> 00:33:15,630 So I'm going to tell you about an approach known as Polymerase 545 00:33:15,630 --> 00:33:19,140 Chain Reaction, or PCR. 546 00:33:19,140 --> 00:33:24,600 And what PCR is, is it's an in vitro method. 547 00:33:24,600 --> 00:33:31,920 So it's an in vitro approach to essentially amplify DNA. 548 00:33:36,180 --> 00:33:38,880 And so let's say you have a piece of DNA-- 549 00:33:38,880 --> 00:33:41,400 it could be a piece of DNA in the genome-- 550 00:33:41,400 --> 00:33:43,230 and you know the sequence of this DNA. 551 00:33:46,180 --> 00:33:51,300 And it has base pairs between the two strands. 552 00:33:51,300 --> 00:33:53,910 So, normally, for DNA replication to occur, 553 00:33:53,910 --> 00:33:56,387 what do you need? 554 00:33:56,387 --> 00:33:57,470 What needs to happen here? 555 00:34:01,340 --> 00:34:04,260 Can a polymerase get in now? 556 00:34:04,260 --> 00:34:04,760 No? 557 00:34:04,760 --> 00:34:05,750 Why not? 558 00:34:05,750 --> 00:34:07,156 Miles? 559 00:34:07,156 --> 00:34:09,621 AUDIENCE: The DNA's going to-- 560 00:34:09,621 --> 00:34:12,579 because they'll try and [INAUDIBLE] away [INAUDIBLE] 561 00:34:12,579 --> 00:34:14,550 from each other, so you have to [INAUDIBLE].. 562 00:34:14,550 --> 00:34:16,550 ADAM MARTIN: Yeah, you have to unwind it, right? 563 00:34:16,550 --> 00:34:20,239 So you have to denature it first. 564 00:34:20,239 --> 00:34:23,510 So if you do nature it, now you have two single-stranded pieces 565 00:34:23,510 --> 00:34:26,510 of DNA, right? 566 00:34:26,510 --> 00:34:31,570 Now what would a polymerase need to replicate that? 567 00:34:31,570 --> 00:34:32,290 Yeah, Jeremy? 568 00:34:32,290 --> 00:34:33,040 AUDIENCE: A prime. 569 00:34:33,040 --> 00:34:33,957 ADAM MARTIN: A primer. 570 00:34:33,957 --> 00:34:35,040 Exactly, right? 571 00:34:35,040 --> 00:34:37,170 And if you know the sequence, you 572 00:34:37,170 --> 00:34:40,590 can have a company synthesize a primer that's 573 00:34:40,590 --> 00:34:44,520 the exact sequence here and base pairs here. 574 00:34:44,520 --> 00:34:46,650 And I'll just draw the five-prime end of the primer 575 00:34:46,650 --> 00:34:47,699 right there. 576 00:34:47,699 --> 00:34:51,840 And now this primer has a free three-prime hydroxyl here. 577 00:34:51,840 --> 00:34:53,639 And if you added a polymerase, it 578 00:34:53,639 --> 00:34:56,820 would synthesize this bottom strand here. 579 00:34:56,820 --> 00:34:58,515 So this is known as the forward primer. 580 00:35:01,720 --> 00:35:03,640 And on the other strand, you can design 581 00:35:03,640 --> 00:35:08,150 a primer that's complementary to these bases here. 582 00:35:08,150 --> 00:35:10,780 Again, the five-primer end is out. 583 00:35:10,780 --> 00:35:13,600 This would be known as the reverse primer. 584 00:35:13,600 --> 00:35:16,480 And then you could have the DNA polymerase 585 00:35:16,480 --> 00:35:18,670 synthesize the opposite strand. 586 00:35:21,640 --> 00:35:24,460 All right, so the step here will be to first denature. 587 00:35:24,460 --> 00:35:28,310 So the first step would be to melt or denature 588 00:35:28,310 --> 00:35:30,355 the DNA, double-stranded DNA. 589 00:35:32,920 --> 00:35:35,630 So you denature the double-stranded DNA. 590 00:35:35,630 --> 00:35:40,870 This is commonly done above 90 degrees Celsius. 591 00:35:40,870 --> 00:35:42,460 And then the next step is once you 592 00:35:42,460 --> 00:35:45,400 have these single-stranded pieces of DNA, 593 00:35:45,400 --> 00:35:49,540 you can act you can have a primer present that anneals 594 00:35:49,540 --> 00:35:51,730 to the opposite strands. 595 00:35:51,730 --> 00:35:53,850 So you can have primer annealing. 596 00:35:59,020 --> 00:36:03,880 And this is commonly done between 50 and 60 degrees 597 00:36:03,880 --> 00:36:04,670 Celsius. 598 00:36:04,670 --> 00:36:08,290 You have to cool it down so that the primer can now base pair, 599 00:36:08,290 --> 00:36:10,600 such that not everything is denatured. 600 00:36:10,600 --> 00:36:12,670 So you have to cool it down for these primers 601 00:36:12,670 --> 00:36:16,030 to recognize their cognate sequence and base pair with it. 602 00:36:19,670 --> 00:36:21,970 And then once you have the primer annealed 603 00:36:21,970 --> 00:36:26,530 to the template, then you can add DNA polymerase 604 00:36:26,530 --> 00:36:29,080 to synthesize a new strand. 605 00:36:29,080 --> 00:36:35,440 So DNA polymerize for new strand synthesis. 606 00:36:35,440 --> 00:36:41,300 And this is commonly done at around 70 degrees Celsius. 607 00:36:41,300 --> 00:36:45,240 And then you can repeat this process over and over again. 608 00:36:45,240 --> 00:36:48,160 And at each step, you're going to double the amount of DNA 609 00:36:48,160 --> 00:36:52,420 that you have between these two primers. 610 00:36:52,420 --> 00:36:55,540 So let me just-- this is just a figure illustrating this. 611 00:36:55,540 --> 00:36:57,940 It's on the handout and online. 612 00:36:57,940 --> 00:37:01,540 Basically, you have your original double-stranded piece 613 00:37:01,540 --> 00:37:02,870 of DNA. 614 00:37:02,870 --> 00:37:05,860 You denature it and allow the primers to anneal. 615 00:37:05,860 --> 00:37:08,320 New strand synthesis. 616 00:37:08,320 --> 00:37:13,260 Then you take these new pieces of double-stranded DNA, 617 00:37:13,260 --> 00:37:14,890 denature them. 618 00:37:14,890 --> 00:37:17,950 The primers anneal to those new strands, 619 00:37:17,950 --> 00:37:19,540 and now you get new strands. 620 00:37:19,540 --> 00:37:22,990 And you just keep doing this cycle over and over again, 621 00:37:22,990 --> 00:37:26,620 and you essentially amplify the piece of DNA 622 00:37:26,620 --> 00:37:30,010 that's between the two primer sequences. 623 00:37:30,010 --> 00:37:31,710 So this is often used in forensics, 624 00:37:31,710 --> 00:37:34,690 because you can have very little DNA, 625 00:37:34,690 --> 00:37:37,930 and just by adding primers, you can really 626 00:37:37,930 --> 00:37:41,080 amplify the number of pieces of DNA 627 00:37:41,080 --> 00:37:43,360 you have between these two fragments. 628 00:37:43,360 --> 00:37:47,980 So you go from having very little DNA to a lot of DNA. 629 00:37:47,980 --> 00:37:51,460 OK, any questions about PCR? 630 00:37:51,460 --> 00:37:55,620 I'm going to move on to something that-- 631 00:37:55,620 --> 00:37:56,120 all right. 632 00:37:59,030 --> 00:38:02,190 I've really been focused on discovery up to this point. 633 00:38:02,190 --> 00:38:05,270 But I know that a number of you are engineers, 634 00:38:05,270 --> 00:38:09,470 and you probably want to engineer something. 635 00:38:09,470 --> 00:38:10,940 And so I've had to-- 636 00:38:14,030 --> 00:38:16,010 I'm going to tell you about a field that 637 00:38:16,010 --> 00:38:19,040 is moving so rapidly, I'm going to probably have 638 00:38:19,040 --> 00:38:22,380 to totally revamp my lecture for next year, OK? 639 00:38:22,380 --> 00:38:26,120 And I'm going to tell you about genome editing. 640 00:38:26,120 --> 00:38:31,250 So the last part of this story, genome or DNA editing. 641 00:38:31,250 --> 00:38:35,420 And I'm going to tell you about a specific type of system 642 00:38:35,420 --> 00:38:39,530 called CRISPR-Cas9, which has been in the news a lot, 643 00:38:39,530 --> 00:38:42,170 and there's a lot of excitement about this approach 644 00:38:42,170 --> 00:38:45,020 in the context of editing the human genome 645 00:38:45,020 --> 00:38:47,870 and possibly curing genetic diseases. 646 00:38:47,870 --> 00:38:50,690 Who here has heard of CRISPR-Cas9? 647 00:38:50,690 --> 00:38:53,040 OK, good. 648 00:38:53,040 --> 00:38:53,540 That's good. 649 00:38:53,540 --> 00:38:54,875 Our media is doing its job. 650 00:38:57,620 --> 00:39:01,080 So who knows what it is? 651 00:39:01,080 --> 00:39:03,120 OK, some of us know what it is. 652 00:39:03,120 --> 00:39:06,600 I just want to just give you a very superficial overview 653 00:39:06,600 --> 00:39:08,735 of what it is and why it's important. 654 00:39:08,735 --> 00:39:10,110 And I'm going to keep coming back 655 00:39:10,110 --> 00:39:12,270 to it during the course of the semester, 656 00:39:12,270 --> 00:39:15,710 because I think it raises a lot of ethical questions, 657 00:39:15,710 --> 00:39:18,120 and especially in the context of stem cells. 658 00:39:18,120 --> 00:39:20,160 I need you to know the foundation before we 659 00:39:20,160 --> 00:39:24,090 get into the really good stuff. 660 00:39:24,090 --> 00:39:26,460 So, let's see. 661 00:39:26,460 --> 00:39:29,220 So we're going to engineer something. 662 00:39:29,220 --> 00:39:32,710 So we're going to talk about repairing DNA. 663 00:39:32,710 --> 00:39:34,860 And if we want to edit the genome, 664 00:39:34,860 --> 00:39:37,410 the way this is most often done is 665 00:39:37,410 --> 00:39:41,040 by making a double-stranded break, OK? 666 00:39:41,040 --> 00:39:45,090 So if you make a double-stranded break in a piece of DNA, 667 00:39:45,090 --> 00:39:47,770 it can be repaired one of two ways. 668 00:39:47,770 --> 00:39:49,770 One is by non-homologous end joining, 669 00:39:49,770 --> 00:39:52,200 where the two pieces of DNA are basically just shoved 670 00:39:52,200 --> 00:39:53,730 back together again. 671 00:39:53,730 --> 00:39:56,370 And this results, often, in mutations. 672 00:39:56,370 --> 00:39:58,117 So if you're trying to fix something, 673 00:39:58,117 --> 00:39:59,700 unless you're just trying to break it, 674 00:39:59,700 --> 00:40:01,770 that's probably not what you want. 675 00:40:01,770 --> 00:40:04,380 But an alternative approach to DNA 676 00:40:04,380 --> 00:40:07,200 repair that organisms have is something called 677 00:40:07,200 --> 00:40:09,800 homology-directed repair. 678 00:40:09,800 --> 00:40:12,660 In this case, you can break a piece of DNA 679 00:40:12,660 --> 00:40:16,170 and add a piece of DNA that has a different sequence, 680 00:40:16,170 --> 00:40:20,350 but with homology near where the double-stranded break is. 681 00:40:20,350 --> 00:40:24,360 And in that case, you can replace the original sequence 682 00:40:24,360 --> 00:40:27,690 with what you provide as donor DNA. 683 00:40:27,690 --> 00:40:30,150 So it gives you an ability to essentially 684 00:40:30,150 --> 00:40:33,630 change the DNA sequence at a given locus 685 00:40:33,630 --> 00:40:39,360 if you're able to cleave the DNA at a specific locus. 686 00:40:39,360 --> 00:40:42,240 So, first, I want to start with just a thought 687 00:40:42,240 --> 00:40:43,470 experiment, right? 688 00:40:43,470 --> 00:40:48,030 You're all thinking, OK, we need to cleave double-stranded DNA. 689 00:40:48,030 --> 00:40:50,820 And boy, I just gave you a perfect tool for that. 690 00:40:50,820 --> 00:40:54,030 I gave you all these restriction endonucleases, right? 691 00:40:54,030 --> 00:40:55,620 So what's the problem with those? 692 00:40:55,620 --> 00:40:58,260 Well, let's think about the human genome. 693 00:40:58,260 --> 00:41:02,490 The human genome is 3 billion base pairs. 694 00:41:05,580 --> 00:41:08,230 And an EcoR1 site looks like this-- 695 00:41:08,230 --> 00:41:08,730 GAATTC. 696 00:41:11,880 --> 00:41:15,000 So it's six nucleotides long. 697 00:41:15,000 --> 00:41:17,790 And if you think of just a random sequence 698 00:41:17,790 --> 00:41:21,780 of 3 billion base pairs, you would 699 00:41:21,780 --> 00:41:25,890 get this sequence randomly one out of every four 700 00:41:25,890 --> 00:41:28,350 to the sixth times. 701 00:41:28,350 --> 00:41:35,010 So that's going to be one every 4,096 times. 702 00:41:35,010 --> 00:41:39,930 So if you get this in random DNA 1 every roughly 4,000 times, 703 00:41:39,930 --> 00:41:42,410 if you use it to cleave the human genome, 704 00:41:42,410 --> 00:41:44,760 it's going to cleave hundreds of thousands 705 00:41:44,760 --> 00:41:47,460 of places in the human genome. 706 00:41:47,460 --> 00:41:50,280 So we need much more specificity if we 707 00:41:50,280 --> 00:41:52,080 want to select, let's say, a given 708 00:41:52,080 --> 00:41:55,800 gene that has a disease-causing allele and try to fix it. 709 00:41:55,800 --> 00:41:58,140 Because if we use a restriction endonuclease, 710 00:41:58,140 --> 00:42:02,650 we just chop up the whole genome, and that would be bad. 711 00:42:02,650 --> 00:42:05,490 So specificity is the name of the game here. 712 00:42:05,490 --> 00:42:09,030 This is not specific, and we need 713 00:42:09,030 --> 00:42:11,790 a tool that's more specific. 714 00:42:11,790 --> 00:42:15,150 And that tool is going to be CRISPR-Cas9. 715 00:42:15,150 --> 00:42:18,870 And what CRISPR-Cas9 is, is it's essentially 716 00:42:18,870 --> 00:42:23,060 an RNA-guided endonuclease. 717 00:42:23,060 --> 00:42:29,070 So it's RNA guided, and it's an endonuclease. 718 00:42:29,070 --> 00:42:32,070 Restriction enzymes, right, they have nothing to do with RNA. 719 00:42:32,070 --> 00:42:36,870 They don't use RNA to recognize the nucleotide sequence. 720 00:42:36,870 --> 00:42:38,490 It's just a protein, and the protein 721 00:42:38,490 --> 00:42:41,070 recognizes the nucleotide sequence. 722 00:42:41,070 --> 00:42:48,015 In CRISPR-Cas9, you have an endonuclease, which is Cas9. 723 00:42:48,015 --> 00:42:51,500 Let's bump this up. 724 00:42:51,500 --> 00:42:53,720 So the endonuclease is the-- 725 00:42:53,720 --> 00:42:55,700 the Cas9 is the protein. 726 00:42:55,700 --> 00:42:57,110 That's the endonuclease. 727 00:43:01,400 --> 00:43:06,440 But its selection of a target depends on an RNA molecule 728 00:43:06,440 --> 00:43:08,600 that it's bound to, OK? 729 00:43:08,600 --> 00:43:14,000 So the specificity comes, at least in part, 730 00:43:14,000 --> 00:43:20,270 from what's known as a guide RNA, or single guide RNA. 731 00:43:20,270 --> 00:43:24,930 That's what's most often used in genome editing. 732 00:43:24,930 --> 00:43:29,180 So this guide RNA basically allows this enzyme 733 00:43:29,180 --> 00:43:32,030 to find a specific sequence. 734 00:43:32,030 --> 00:43:34,730 And the guide RNA is 20 nucleotides, 735 00:43:34,730 --> 00:43:38,420 or looks for homology for a 20-nucleotide base pair 736 00:43:38,420 --> 00:43:39,620 sequence. 737 00:43:39,620 --> 00:43:43,040 So you can see, already, we're doing way better than the six 738 00:43:43,040 --> 00:43:45,140 base pair recognition motif. 739 00:43:45,140 --> 00:43:46,910 We have 20 nucleotides. 740 00:43:46,910 --> 00:43:49,040 And there are other components of the system 741 00:43:49,040 --> 00:43:53,000 which increase the specificity. 742 00:43:53,000 --> 00:43:55,220 Then you have your Cas9 in blue, which 743 00:43:55,220 --> 00:43:59,350 is the endonuclease, your RNA, the guide RNA, in black, 744 00:43:59,350 --> 00:44:01,640 and the template here is in gray. 745 00:44:01,640 --> 00:44:03,680 And what you see is this RNA sort 746 00:44:03,680 --> 00:44:08,570 of exhibiting complementarity to this target sequence. 747 00:44:08,570 --> 00:44:12,260 And only if there's complementarity between the RNA 748 00:44:12,260 --> 00:44:15,140 and the target will this endonuclease 749 00:44:15,140 --> 00:44:18,800 get activated and cleave at this site. 750 00:44:18,800 --> 00:44:22,220 So the RNA is sort of serving like a guide dog 751 00:44:22,220 --> 00:44:27,345 for this endonuclease to guide it to a certain location 752 00:44:27,345 --> 00:44:27,845 to cleave. 753 00:44:34,370 --> 00:44:37,640 So the idea, then, is if you want to edit the genome-- 754 00:44:37,640 --> 00:44:40,760 and why people are so excited about this 755 00:44:40,760 --> 00:44:44,240 these days is you now have a system that 756 00:44:44,240 --> 00:44:48,710 might allow you to generate a double-stranded break in one 757 00:44:48,710 --> 00:44:51,230 specific place in the genome. 758 00:44:51,230 --> 00:44:55,880 And if you can do that, then if you provide donor DNA that 759 00:44:55,880 --> 00:44:59,060 maybe has a different sequence-- if you consider a disease 760 00:44:59,060 --> 00:44:59,870 allele, right? 761 00:44:59,870 --> 00:45:03,440 Let's say you know there's a gene that when 762 00:45:03,440 --> 00:45:08,390 there's a certain allele causes an inherited form of a disease. 763 00:45:08,390 --> 00:45:13,340 You could then take donor DNA from an unaffected individual 764 00:45:13,340 --> 00:45:16,730 and take cells from the affected individual 765 00:45:16,730 --> 00:45:19,790 and cut the locus that's problematic 766 00:45:19,790 --> 00:45:25,220 and get a repair of the defective allele using 767 00:45:25,220 --> 00:45:27,470 a normal allele of the gene. 768 00:45:27,470 --> 00:45:29,390 And that would essentially rescue 769 00:45:29,390 --> 00:45:32,030 the function of that gene if it were then reintroduced 770 00:45:32,030 --> 00:45:33,655 into the patient, OK? 771 00:45:33,655 --> 00:45:38,270 Do you see sort of roughly how this works? 772 00:45:38,270 --> 00:45:41,960 So this is a very sort of broad and general sort 773 00:45:41,960 --> 00:45:44,810 of conceptual framework for how this might happen. 774 00:45:44,810 --> 00:45:47,900 Let's say you have an individual with a blood disorder-- 775 00:45:47,900 --> 00:45:51,350 let's say sickle cell anemia or beta thalassemia. 776 00:45:51,350 --> 00:45:53,700 Those are inherited blood disorders, 777 00:45:53,700 --> 00:45:55,730 which lead to anemia. 778 00:45:55,730 --> 00:45:59,600 You could remove cells, and what might be the best 779 00:45:59,600 --> 00:46:02,840 are the stem cells from a patient. 780 00:46:02,840 --> 00:46:05,420 And you could then take those stem cells and use 781 00:46:05,420 --> 00:46:09,410 CRISPR-Cas9 in vitro in cell culture 782 00:46:09,410 --> 00:46:14,420 to edit that individual's cells to repair the genetic defect. 783 00:46:14,420 --> 00:46:16,700 And you could then reintroduce those 784 00:46:16,700 --> 00:46:19,910 to the patient, where if they're stem cells, 785 00:46:19,910 --> 00:46:22,520 they'd reoccupy the stem cell niche 786 00:46:22,520 --> 00:46:24,530 and produce functional blood cells that 787 00:46:24,530 --> 00:46:27,810 would then essentially cure the individual of the disease. 788 00:46:27,810 --> 00:46:29,330 This is how scientists are thinking 789 00:46:29,330 --> 00:46:32,380 about the use of the system nowadays. 790 00:46:32,380 --> 00:46:35,090 And this hasn't really been successful yet, 791 00:46:35,090 --> 00:46:37,800 but there are several clinical trials that are currently 792 00:46:37,800 --> 00:46:41,990 underway, where people are trying 793 00:46:41,990 --> 00:46:47,390 to show that this can be used to treat human genetic diseases. 794 00:46:47,390 --> 00:46:50,360 So in the next year, you are going to hear more about this, 795 00:46:50,360 --> 00:46:55,040 almost undoubtedly, as we start to hear the results of some 796 00:46:55,040 --> 00:46:56,000 of these patients. 797 00:46:56,000 --> 00:46:58,290 There are concerns about this, as well. 798 00:46:58,290 --> 00:47:00,500 I don't want to overblow it. 799 00:47:00,500 --> 00:47:01,730 There are certainly concerns. 800 00:47:01,730 --> 00:47:03,560 We don't know this is going to work. 801 00:47:03,560 --> 00:47:06,020 I mean, people have been talking about this type of stuff 802 00:47:06,020 --> 00:47:08,570 since I was a student 20 years ago. 803 00:47:08,570 --> 00:47:11,900 But I feel like we're getting-- we're much more advanced now, 804 00:47:11,900 --> 00:47:13,520 and the tools are more advanced. 805 00:47:13,520 --> 00:47:17,300 And so I feel like we're kind of getting to the point where 806 00:47:17,300 --> 00:47:19,910 there's a much greater chance that this will be successful 807 00:47:19,910 --> 00:47:23,370 these days than it was 20 years ago. 808 00:47:23,370 --> 00:47:25,400 I just want to point out where this system-- 809 00:47:25,400 --> 00:47:28,790 how it was discovered and where it came from. 810 00:47:28,790 --> 00:47:30,440 And I like this as an example. 811 00:47:30,440 --> 00:47:34,280 Much like for the fly genes that defined 812 00:47:34,280 --> 00:47:37,370 major signaling pathways, this is a discovery 813 00:47:37,370 --> 00:47:40,760 that came from fundamental research 814 00:47:40,760 --> 00:47:46,100 on, basically, the ecology of bacteria. 815 00:47:46,100 --> 00:47:50,060 So this CRISPR-Cas9 system essentially evolved 816 00:47:50,060 --> 00:47:54,920 in bacteria as a form of an arms race between bacteria 817 00:47:54,920 --> 00:47:57,260 and their predators, bacteriophage, 818 00:47:57,260 --> 00:48:00,900 which are viruses that infect bacteria. 819 00:48:00,900 --> 00:48:09,410 So this is an arms race between bacteria 820 00:48:09,410 --> 00:48:12,020 and their vicious predators, bacteriophage. 821 00:48:18,560 --> 00:48:22,400 And what CRISPR is, where these enzymes and this system 822 00:48:22,400 --> 00:48:27,260 evolved from, is this is a form of an adaptive immune system 823 00:48:27,260 --> 00:48:31,310 for bacteria, which is pretty wild in and of itself. 824 00:48:31,310 --> 00:48:36,965 So CRISPR is an adaptive immune system for bacteria. 825 00:48:41,870 --> 00:48:45,170 If you haven't gotten your flu shot, you should get it. 826 00:48:45,170 --> 00:48:50,580 We'll talk about human immunity later in the semester. 827 00:48:50,580 --> 00:48:54,550 But this is where bacterial immunity kind of-- 828 00:48:54,550 --> 00:48:56,570 I'm sneaking it in. 829 00:48:56,570 --> 00:48:59,910 So the way this works in bacteria-- 830 00:48:59,910 --> 00:49:03,110 what CRISPR stands for is Clusters 831 00:49:03,110 --> 00:49:07,710 of Regularly Interspaced Short Palindromic Repeats. 832 00:49:07,710 --> 00:49:11,060 So you can see already thank god they gave it an acronym. 833 00:49:11,060 --> 00:49:14,160 Otherwise, it wouldn't be getting nearly as much buzz, 834 00:49:14,160 --> 00:49:16,340 because no one can say that. 835 00:49:16,340 --> 00:49:20,270 And so where this CRISPR came from is 836 00:49:20,270 --> 00:49:24,660 on bacterial chromosomes of many bacteria, 837 00:49:24,660 --> 00:49:28,610 there's these clusters of interspaced short palindromic 838 00:49:28,610 --> 00:49:33,240 repeats, and the repeats are interrupted by spacers. 839 00:49:33,240 --> 00:49:36,780 And what researchers discovered are these spacers 840 00:49:36,780 --> 00:49:41,190 have sequence similarity and identity to sequences 841 00:49:41,190 --> 00:49:44,970 that are from bacteriophage. 842 00:49:44,970 --> 00:49:46,920 So each of these colored sequences 843 00:49:46,920 --> 00:49:51,000 here has some type of complementarity 844 00:49:51,000 --> 00:49:54,510 to some type of bacteriophage. 845 00:49:54,510 --> 00:49:59,310 And so when a phage infects bacteria, or some bacteria, 846 00:49:59,310 --> 00:50:01,890 what happens is that there's a system 847 00:50:01,890 --> 00:50:05,370 to recognize that foreign genetic element 848 00:50:05,370 --> 00:50:09,160 and take a piece of it and insert it in the genome. 849 00:50:09,160 --> 00:50:11,190 And that serves as a memory for the bacteria 850 00:50:11,190 --> 00:50:16,200 to remember that it got infected by that particular phage. 851 00:50:16,200 --> 00:50:18,000 And then, later on, what the bacteria 852 00:50:18,000 --> 00:50:21,570 does is it transcribes this region 853 00:50:21,570 --> 00:50:25,950 and forms these mature what are known as CRISPR RNAs, where you 854 00:50:25,950 --> 00:50:28,920 can see there's some sequence would recognize 855 00:50:28,920 --> 00:50:31,210 a foreign genetic element. 856 00:50:31,210 --> 00:50:36,030 So, therefore, in the future, if this phage came around again, 857 00:50:36,030 --> 00:50:39,450 what would happen is one of these CRISPR RNAs 858 00:50:39,450 --> 00:50:42,450 would recognize the foreign genetic element 859 00:50:42,450 --> 00:50:45,030 through base pair complementarity, 860 00:50:45,030 --> 00:50:46,830 and it would know to cut it. 861 00:50:46,830 --> 00:50:49,050 And after the target is cut, it's 862 00:50:49,050 --> 00:50:51,930 then degraded by the bacterial cell. 863 00:50:51,930 --> 00:50:56,220 So this is a way for bacteria to remember what viruses 864 00:50:56,220 --> 00:51:01,260 have infected them and to have a defense mechanism against it. 865 00:51:01,260 --> 00:51:03,810 So it's a pretty cool system. 866 00:51:03,810 --> 00:51:05,670 You know, what's also cool about this system 867 00:51:05,670 --> 00:51:09,720 is it's an adaptive immune system, similar to how we sort 868 00:51:09,720 --> 00:51:13,140 of recognize foreign pathogens. 869 00:51:13,140 --> 00:51:15,810 What's different about it is this is heritable. 870 00:51:15,810 --> 00:51:18,120 It's incorporated into the genome. 871 00:51:18,120 --> 00:51:21,300 And the more phage the descendants of this bacteria 872 00:51:21,300 --> 00:51:24,510 see, the more of these repeats you see. 873 00:51:24,510 --> 00:51:28,080 So this is a heritable immune system, which, unfortunately, 874 00:51:28,080 --> 00:51:30,630 we don't have. 875 00:51:30,630 --> 00:51:32,280 So you should still get your flu shot. 876 00:51:35,970 --> 00:51:38,010 We'll talk about vaccination later on. 877 00:51:40,680 --> 00:51:44,480 Have a good few days, and I will see you on Friday.