1 00:00:06,710 --> 00:00:10,770 PROFESSOR: Now having cut DNA, we then need to 2 00:00:10,770 --> 00:00:12,020 do something else. 3 00:00:14,290 --> 00:00:18,245 What's the next thing we have to do? 4 00:00:18,245 --> 00:00:19,710 AUDIENCE: Paste it. 5 00:00:19,710 --> 00:00:21,290 PROFESSOR: Paste it right. 6 00:00:21,290 --> 00:00:23,601 Oh, I put past. 7 00:00:23,601 --> 00:00:25,840 It should be paste. 8 00:00:25,840 --> 00:00:26,890 There we go. 9 00:00:26,890 --> 00:00:28,680 We should paste it. 10 00:00:28,680 --> 00:00:32,780 So, how are we going to paste our DNA? 11 00:00:32,780 --> 00:00:34,960 So let's take some human DNA. 12 00:00:39,020 --> 00:00:41,360 We'll take your human DNA. 13 00:00:41,360 --> 00:00:43,490 We'll add EcoR1 to it. 14 00:00:43,490 --> 00:00:48,870 And your human DNA is going to get cut up in lots of little 15 00:00:48,870 --> 00:00:52,680 pieces of length about 4,000. 16 00:00:52,680 --> 00:00:56,420 And I'm writing R1 at the ends because these pieces of DNA 17 00:00:56,420 --> 00:00:59,230 have EcoR1 sites. 18 00:00:59,230 --> 00:01:05,790 Now, I can take that human DNA and I can combine it with 19 00:01:05,790 --> 00:01:07,040 other pieces of DNA. 20 00:01:11,270 --> 00:01:12,710 Here's a piece of DNA. 21 00:01:12,710 --> 00:01:16,500 Remember it had this overhang like that. 22 00:01:19,900 --> 00:01:25,410 T-T-A-A was this piece here of human DNA. 23 00:01:29,420 --> 00:01:34,510 And I could take another piece of DNA that matched it a 24 00:01:34,510 --> 00:01:41,100 A-A-T-T. 25 00:01:41,100 --> 00:01:42,290 And it doesn't have to be human. 26 00:01:42,290 --> 00:01:44,080 It could be something else. 27 00:01:44,080 --> 00:01:45,330 It could be zebra. 28 00:01:47,660 --> 00:01:53,320 Some human DNA, some zebra DNA, and by that base pairing 29 00:01:53,320 --> 00:01:57,732 of those four bases, they'll sort of stick. 30 00:01:57,732 --> 00:01:58,580 It's not that strong. 31 00:01:58,580 --> 00:02:01,536 It's four bases of base pairing, but they'll stick. 32 00:02:01,536 --> 00:02:05,100 Now I'd like to glue them together. 33 00:02:05,100 --> 00:02:08,889 What's the word for attaching together DNA strands? 34 00:02:08,889 --> 00:02:10,500 We talked about it-- 35 00:02:10,500 --> 00:02:15,010 Ligate, we want to ligate them together. 36 00:02:15,010 --> 00:02:17,750 So now we go back to our MIT engineers and say, please 37 00:02:17,750 --> 00:02:20,150 invent me a protein that's able to ligate 38 00:02:20,150 --> 00:02:22,850 together pieces of DNA. 39 00:02:22,850 --> 00:02:26,720 But actually, who invented it first? 40 00:02:26,720 --> 00:02:28,220 Bacteria invented it first. 41 00:02:28,220 --> 00:02:30,260 And it's called? 42 00:02:30,260 --> 00:02:31,620 Ligase. 43 00:02:31,620 --> 00:02:34,110 So all we have to do is add ligase. 44 00:02:34,110 --> 00:02:35,450 So it was kind of useful to know how 45 00:02:35,450 --> 00:02:37,180 DNA replication worked. 46 00:02:37,180 --> 00:02:40,110 We add ligase. 47 00:02:40,110 --> 00:02:44,260 And what does ligase do for a living? 48 00:02:44,260 --> 00:02:48,230 For a living, ligase is paid to go around and find pieces 49 00:02:48,230 --> 00:02:52,890 of DNA that have nicks and seal them back up by repairing 50 00:02:52,890 --> 00:02:54,860 the sugar phosphate backbone of DNA. 51 00:02:58,480 --> 00:03:03,700 And again, ancient molecular biologists would prove their 52 00:03:03,700 --> 00:03:08,090 mettle by purifying ligase and using it in their reactions. 53 00:03:08,090 --> 00:03:11,480 And today, where do we get ligase? 54 00:03:11,480 --> 00:03:13,100 It's in the catalog. 55 00:03:13,100 --> 00:03:16,760 Ligase is in the catalog, reasonably cheap. 56 00:03:16,760 --> 00:03:24,770 So, when we add ligase we could now get a hybrid piece 57 00:03:24,770 --> 00:03:28,310 of DNA that was half human and half zebra. 58 00:03:28,310 --> 00:03:30,950 I got to say, this freaks some people out. 59 00:03:30,950 --> 00:03:34,610 This doesn't freak me out because it's just a sequence 60 00:03:34,610 --> 00:03:35,410 of nucleotides. 61 00:03:35,410 --> 00:03:37,920 If I give you a sequence of nucleotides, to my mind, 62 00:03:37,920 --> 00:03:40,990 there's nothing human or zebra about it. 63 00:03:40,990 --> 00:03:42,390 It's a chemical. 64 00:03:42,390 --> 00:03:46,210 And you can make bonds between chemicals and all that. 65 00:03:46,210 --> 00:03:52,080 So, we now pasted our DNA using ligase. 66 00:03:52,080 --> 00:03:54,070 Ligase from the catalog. 67 00:03:54,070 --> 00:03:58,550 Now the question is, what are we going to paste our DNA to? 68 00:04:01,400 --> 00:04:03,185 What do we paste our DNA to? 69 00:04:13,840 --> 00:04:20,910 We're going to paste our DNA to a fascinating other piece 70 00:04:20,910 --> 00:04:29,224 of DNA like this. 71 00:04:29,224 --> 00:04:33,400 I'll make that a little bigger here. 72 00:04:33,400 --> 00:04:38,580 That has an EcoR1 site, has an EcoR1 site. 73 00:04:38,580 --> 00:04:48,680 And we're going to take our human DNA and paste it in such 74 00:04:48,680 --> 00:04:50,890 a way that it makes a circle. 75 00:04:50,890 --> 00:04:55,900 This piece of DNA is a vector. 76 00:04:55,900 --> 00:04:57,470 Vector means it travels around. 77 00:04:57,470 --> 00:04:59,700 It carries things with it. 78 00:04:59,700 --> 00:05:11,840 So I want this vector to be able to replicate if I 79 00:05:11,840 --> 00:05:13,990 transfer it to E Coli. 80 00:05:13,990 --> 00:05:14,810 So that's the trick. 81 00:05:14,810 --> 00:05:17,250 I would like to be able to attachment my human DNA to 82 00:05:17,250 --> 00:05:20,720 this vector DNA, and when I transfer it to E coli, have 83 00:05:20,720 --> 00:05:22,450 this thing be able to grow. 84 00:05:22,450 --> 00:05:27,460 So that means we need to invent a piece of DNA, a 85 00:05:27,460 --> 00:05:32,010 vector, that is capable of causing E. coli 86 00:05:32,010 --> 00:05:34,450 to replicate itself. 87 00:05:34,450 --> 00:05:38,510 It's got to have all the instructions to cause E. coli 88 00:05:38,510 --> 00:05:42,280 to replicate this piece of DNA. 89 00:05:42,280 --> 00:05:46,920 So this is another amazing bit of engineering. 90 00:05:46,920 --> 00:05:51,650 How do we invent just the right sequence of letters that 91 00:05:51,650 --> 00:05:53,990 would allow E. coli to replicate this thing? 92 00:05:53,990 --> 00:05:55,750 AUDIENCE: Already been invented. 93 00:05:55,750 --> 00:05:56,630 PROFESSOR: Sorry. 94 00:05:56,630 --> 00:05:59,030 AUDIENCE: It's already been invented by E. coli. 95 00:05:59,030 --> 00:06:01,490 PROFESSOR: It's already been invented by E. 96 00:06:01,490 --> 00:06:03,610 coli, hasn't it? 97 00:06:03,610 --> 00:06:07,790 So you're getting the theme here, is that we don't 98 00:06:07,790 --> 00:06:10,010 actually do anything in molecular biology. 99 00:06:13,480 --> 00:06:16,830 Now look, in fairness, life's had 3.5 billion years. 100 00:06:16,830 --> 00:06:19,340 We've been at this a decade or two, on the 101 00:06:19,340 --> 00:06:20,790 whole, maybe three. 102 00:06:20,790 --> 00:06:23,080 On the whole, life's had a lot more time to 103 00:06:23,080 --> 00:06:24,090 work this stuff out. 104 00:06:24,090 --> 00:06:27,360 And usually the best solution is to look in nature to see 105 00:06:27,360 --> 00:06:30,420 where nature has already done it for you. 106 00:06:30,420 --> 00:06:35,670 So E. coli, it turns out, replicates its own chromosome. 107 00:06:35,670 --> 00:06:39,010 You could use the machinery from its own chromosome. 108 00:06:39,010 --> 00:06:41,130 But it turns out even better than that. 109 00:06:41,130 --> 00:06:45,230 E. coli has the following. 110 00:06:48,710 --> 00:06:51,570 Here's its big chromosome, four million letters of E. 111 00:06:51,570 --> 00:06:53,790 coli chromosome. 112 00:06:53,790 --> 00:07:00,100 E. Coli also has within it little circles 113 00:07:00,100 --> 00:07:03,080 of DNA called plasmids. 114 00:07:07,590 --> 00:07:10,370 These little circles of DNA are able to 115 00:07:10,370 --> 00:07:12,020 autonomously replicate. 116 00:07:12,020 --> 00:07:13,330 They can copy themselves. 117 00:07:13,330 --> 00:07:17,180 Or that is to say, E. coli copies them. 118 00:07:17,180 --> 00:07:21,310 These little plasmids have the full replication instructions 119 00:07:21,310 --> 00:07:22,480 encoded in them. 120 00:07:22,480 --> 00:07:24,450 Now why does E. coli have these plasmids? 121 00:07:24,450 --> 00:07:25,530 What's in these plasmids? 122 00:07:25,530 --> 00:07:27,220 What's going on? 123 00:07:27,220 --> 00:07:29,050 Well let's blow up one of these plasmids. 124 00:07:31,860 --> 00:07:34,370 It's a big circle. 125 00:07:34,370 --> 00:07:40,960 And it has an instruction here, a sequence, called an 126 00:07:40,960 --> 00:07:51,195 origin of replication - or amongst friends, just ORI. 127 00:07:51,195 --> 00:07:52,480 It's called ORI. 128 00:07:52,480 --> 00:07:55,850 You'll find on maps, origin of replication. 129 00:07:55,850 --> 00:07:59,820 That sequence alone is enough to cause E. coli to open it up 130 00:07:59,820 --> 00:08:04,330 and start doing its DNA replication from the origin. 131 00:08:04,330 --> 00:08:09,520 But what tells you why this is so important is that these 132 00:08:09,520 --> 00:08:16,160 plasmids typically have one or two genes. 133 00:08:16,160 --> 00:08:21,150 And the one or two genes that they typically have are genes 134 00:08:21,150 --> 00:08:29,330 that encode a protein that gives them resistance to an 135 00:08:29,330 --> 00:08:35,424 antibiotic, like say, penicillin. 136 00:08:39,750 --> 00:08:42,200 There could be a gene that gives you penicillin 137 00:08:42,200 --> 00:08:43,450 resistance. 138 00:08:46,070 --> 00:08:47,070 That's kind of cool. 139 00:08:47,070 --> 00:08:49,140 E. coli, or other bacteria-- 140 00:08:49,140 --> 00:08:51,230 not E. coli necessarily for penicillin-- 141 00:08:51,230 --> 00:08:54,520 carry around often little circles. 142 00:08:54,520 --> 00:08:56,500 And these little circles encode genes that give them 143 00:08:56,500 --> 00:09:01,540 resistance to streptomycin, penicillin, ampicillin, all 144 00:09:01,540 --> 00:09:02,790 sorts of things. 145 00:09:06,370 --> 00:09:10,360 This allows it to grow, even if you're taking penicillin. 146 00:09:13,450 --> 00:09:15,840 Now, that's pretty clever. 147 00:09:15,840 --> 00:09:18,090 How did E. Coli come up? 148 00:09:18,090 --> 00:09:21,980 How is it so smart to know to have come up with a gene able 149 00:09:21,980 --> 00:09:24,670 to break down penicillin, given that we've only been 150 00:09:24,670 --> 00:09:29,620 using penicillin since the 1940s? 151 00:09:29,620 --> 00:09:32,510 Pretty fast for E. coli to have figured out how to do 152 00:09:32,510 --> 00:09:33,760 that, isn't it? 153 00:09:39,020 --> 00:09:42,900 Oh yeah, who invented penicillin? 154 00:09:42,900 --> 00:09:44,440 Nature invented penicillin. 155 00:09:44,440 --> 00:09:46,780 It's produced by fungi. 156 00:09:46,780 --> 00:09:50,250 Fungi have been fighting E. Coli with penicillin for 157 00:09:50,250 --> 00:09:52,070 millions of years. 158 00:09:52,070 --> 00:09:55,185 E. coli didn't invent it for us. 159 00:09:55,185 --> 00:09:58,690 E. Coli invented it because it's in a war down there at 160 00:09:58,690 --> 00:10:01,460 the single cell level with fungi. 161 00:10:01,460 --> 00:10:02,850 Fungi make antibi-- 162 00:10:02,850 --> 00:10:04,730 You see, we think we're so cool. 163 00:10:04,730 --> 00:10:06,160 We make antibiotics. 164 00:10:06,160 --> 00:10:07,500 Well we've been making antibiotics 165 00:10:07,500 --> 00:10:09,640 only since the 1940s. 166 00:10:09,640 --> 00:10:12,350 Nature's been making antibiotics forever. 167 00:10:12,350 --> 00:10:15,270 Just like the viruses are infecting E. coli and E. coli 168 00:10:15,270 --> 00:10:17,770 needs an immune system against the viruses. 169 00:10:17,770 --> 00:10:21,150 Well the fungi are throwing antibiotics at the bacteria 170 00:10:21,150 --> 00:10:22,620 and it needs a defense mechanism. 171 00:10:22,620 --> 00:10:25,570 And the defense mechanism are these genes that can break 172 00:10:25,570 --> 00:10:28,140 down those antibiotics. 173 00:10:28,140 --> 00:10:30,040 And as usual, we just come along and we 174 00:10:30,040 --> 00:10:31,860 say, oh look at that. 175 00:10:31,860 --> 00:10:33,020 It's already been invented. 176 00:10:33,020 --> 00:10:33,610 Kind of cool. 177 00:10:33,610 --> 00:10:36,870 We'll use that one too. 178 00:10:36,870 --> 00:10:38,370 Now why are these on these little circles? 179 00:10:38,370 --> 00:10:42,750 They're on the little circles because when this bacteria has 180 00:10:42,750 --> 00:10:47,420 lived a long and happy life and it dies and it spews its 181 00:10:47,420 --> 00:10:56,540 guts out, neighboring bacteria suck up the DNA. 182 00:10:56,540 --> 00:10:59,670 Sounds a little cannibalistic or something. 183 00:10:59,670 --> 00:11:01,280 But that's how it is down there at 184 00:11:01,280 --> 00:11:02,880 the single cell level. 185 00:11:02,880 --> 00:11:05,420 The neighboring bacteria will suck up DNA. 186 00:11:05,420 --> 00:11:06,980 And why is that cool? 187 00:11:06,980 --> 00:11:11,310 It's cool because they can acquire these resistances. 188 00:11:11,310 --> 00:11:12,250 That's pretty impressive. 189 00:11:12,250 --> 00:11:13,670 They can acquire the resistance. 190 00:11:13,670 --> 00:11:16,890 And in fact, it even works across species. 191 00:11:16,890 --> 00:11:18,490 That's why you don't put it on the chromosome here. 192 00:11:18,490 --> 00:11:21,140 Because E. coli could pick it up from another species that's 193 00:11:21,140 --> 00:11:23,160 not E. coli, but related enough that it 194 00:11:23,160 --> 00:11:24,160 can use that plasmid. 195 00:11:24,160 --> 00:11:28,640 And you can transfer antibiotic resistance across 196 00:11:28,640 --> 00:11:32,410 many species of bacteria. 197 00:11:32,410 --> 00:11:36,520 So the bacteria like to suck up DNA and see what they find. 198 00:11:36,520 --> 00:11:38,560 Pretty cool. 199 00:11:38,560 --> 00:11:41,070 By the way, this is also why indiscriminate use of 200 00:11:41,070 --> 00:11:44,560 antibiotics, for example, in animal feed, or when you were 201 00:11:44,560 --> 00:11:47,670 given antibiotics by the MIT health service and are told to 202 00:11:47,670 --> 00:11:50,690 take them for two weeks and you take them for five days, 203 00:11:50,690 --> 00:11:52,260 you're not doing anything very good. 204 00:11:52,260 --> 00:11:55,980 You're just selecting for bacteria that can grow in the 205 00:11:55,980 --> 00:11:59,210 presence of antibiotics and selecting for multidrug 206 00:11:59,210 --> 00:12:01,470 resistance in the spread of antibiotics. 207 00:12:01,470 --> 00:12:02,410 We have to be pretty careful. 208 00:12:02,410 --> 00:12:05,560 Because all these mechanisms of swapping antibiotic 209 00:12:05,560 --> 00:12:08,460 resistance are pretty impressive tricks. 210 00:12:08,460 --> 00:12:10,320 Anyway, so these things exist. 211 00:12:10,320 --> 00:12:12,570 And originally, molecular biologists 212 00:12:12,570 --> 00:12:14,050 discovered these plasmids. 213 00:12:14,050 --> 00:12:15,680 They purified these plasmids. 214 00:12:15,680 --> 00:12:17,460 And the deal was this, they cut the 215 00:12:17,460 --> 00:12:22,620 plasmid with EcoR1, ideally. 216 00:12:22,620 --> 00:12:25,980 So I'm going to put this up here, vectors. 217 00:12:25,980 --> 00:12:27,340 They cut the plasmid with EcoR1. 218 00:12:31,800 --> 00:12:37,060 They ligate human DNA into it. 219 00:12:37,060 --> 00:12:38,640 And they get these circles. 220 00:12:38,640 --> 00:12:41,640 And there you go. 221 00:12:41,640 --> 00:12:48,200 Now today, obviously, if I wanted to do this experiment, 222 00:12:48,200 --> 00:12:52,160 would I purify the plasmid myself? 223 00:12:52,160 --> 00:12:52,540 No. 224 00:12:52,540 --> 00:12:54,100 Where would I get the plasmid from? 225 00:12:54,100 --> 00:12:54,920 AUDIENCE: Catalog. 226 00:12:54,920 --> 00:12:55,980 PROFESSOR: It's in the catalog. 227 00:12:55,980 --> 00:12:57,660 There's a whole section a plasmids here. 228 00:12:57,660 --> 00:12:59,240 They have vectors of all sorts. 229 00:12:59,240 --> 00:13:01,310 So you get it from the catalog. 230 00:13:01,310 --> 00:13:02,560 All right.