1 00:00:08,176 --> 00:00:09,106 >> Dude: Grrr! 2 00:00:09,306 --> 00:00:11,586 Something is really wrong with this! 3 00:00:11,586 --> 00:00:12,916 >> Sally: What are you playing with? 4 00:00:12,916 --> 00:00:15,126 >> Dude: I downloaded an app called "Part Palette." 5 00:00:15,126 --> 00:00:18,406 It lets me plan genetic circuits using Parts from the Registry. 6 00:00:18,526 --> 00:00:19,476 >> Sally: That sounds great. 7 00:00:19,476 --> 00:00:19,996 Can I see? 8 00:00:19,996 --> 00:00:22,926 >> Dude: I can select parts and then wrap them together on the palette. 9 00:00:22,926 --> 00:00:24,866 Or at least I'm suppose to be able to do that! 10 00:00:24,866 --> 00:00:24,356 I keep getting an error message. 11 00:00:24,406 --> 00:00:24,856 >> Sally: Hmmm... 12 00:00:24,976 --> 00:00:29,326 maybe the app doesn't work, or maybe there's something 13 00:00:29,326 --> 00:00:31,126 about the parts you're trying to wrap together. 14 00:00:31,436 --> 00:00:32,796 What gene are you trying to build? 15 00:00:32,796 --> 00:00:34,406 >> Dude: I just grabbed these 3 parts to try. 16 00:00:34,596 --> 00:00:38,076 >> Sally: A terminator, an operator and an open reading frame? 17 00:00:38,076 --> 00:00:39,366 That's not a gene... 18 00:00:39,366 --> 00:00:42,616 >> Dude: but it's got an open reading frame...what more do I need? 19 00:00:42,616 --> 00:00:43,476 >> Sally: Walk with me Dude. 20 00:00:43,596 --> 00:00:45,356 For starters you'll need a promoter. 21 00:00:45,616 --> 00:00:50,766 That's a snippet of DNA that the RNA polymerase binds to when it starts transcription. 22 00:00:51,146 --> 00:00:54,046 Promoters come in different strengths so choose carefully. 23 00:00:54,046 --> 00:00:56,446 >> Dude: How do I know if a promoter is strong or weak? 24 00:00:56,446 --> 00:00:59,446 >> Sally: Well the strongest promoters have this DNA sequence. 25 00:00:59,526 --> 00:01:02,046 RNA polymerase can bind these bases well. 26 00:01:02,046 --> 00:01:06,606 The more bases that are different from this consensus sequence, the weaker the promoter is 27 00:01:06,606 --> 00:01:11,576 since RNA polymerase will bind less well and transcribe less often...at least... 28 00:01:11,626 --> 00:01:13,286 that's how it usually works. 29 00:01:13,286 --> 00:01:13,376 >> Dude: oh no! 30 00:01:13,376 --> 00:01:15,386 I hear an exception coming... 31 00:01:15,386 --> 00:01:17,606 >> Sally: Well sure Dude...it's very clever really. 32 00:01:17,876 --> 00:01:20,816 There are promoters that are used in different stages of cell growth 33 00:01:20,816 --> 00:01:23,816 and those promoters bind a slightly different RNA polymerase 34 00:01:23,816 --> 00:01:26,816 so they have a consensus sequence that's a little different from this one. 35 00:01:26,816 --> 00:01:30,036 And then there are still other promoters that have lots of differences 36 00:01:30,036 --> 00:01:33,086 from this consensus sequence but they are still strong promoters 37 00:01:33,086 --> 00:01:37,096 since they also have DNA sequences nearby that bind activating proteins. 38 00:01:37,456 --> 00:01:40,186 The activators help RNA polymerase find the promoter 39 00:01:40,226 --> 00:01:42,246 and that gets transcription really cranking. 40 00:01:42,246 --> 00:01:44,366 Activated promoters can be very strong. 41 00:01:44,426 --> 00:01:45,866 >> Dude: When does it end!?! 42 00:01:45,936 --> 00:01:48,996 >> Sally: Another great question Dude!...there are sequences that come 43 00:01:48,996 --> 00:01:51,076 after the open reading frame called terminators. 44 00:01:51,186 --> 00:01:55,346 These tell RNA polymerase to stop transcribing RNA and to leave the DNA. 45 00:01:55,346 --> 00:01:59,956 These terminators are really useful if you have another promoter downstream of this gene. 46 00:02:00,296 --> 00:02:02,986 You'll need a terminator if you don't want the polymerase to read 47 00:02:02,986 --> 00:02:05,166 through this gene into the next one downstream. 48 00:02:05,236 --> 00:02:06,226 >> Dude: "Downstream?" 49 00:02:06,466 --> 00:02:08,515 Don't you mean "on the right?" 50 00:02:08,596 --> 00:02:10,826 >> Sally: Cells don't know their right from their left so we have 51 00:02:10,826 --> 00:02:14,566 to use the natural polarity of double stranded DNA when we give directions. 52 00:02:14,756 --> 00:02:18,776 "Downstream" means "more 3-prime" no matter which strand of the DNA you're working on. 53 00:02:18,776 --> 00:02:22,056 >> Dude: So in this app I can wrap any promoter, 54 00:02:22,056 --> 00:02:26,806 ORF and terminator together, and I've got a gene! 55 00:02:26,806 --> 00:02:28,816 >> Sally: Well actually you've got a transcription unit, 56 00:02:28,816 --> 00:02:32,746 and most of the time you'll want to translate the RNA from that unit into a protein. 57 00:02:32,746 --> 00:02:36,156 To translate it, you'll need to include a ribosome binding site. 58 00:02:36,156 --> 00:02:39,076 The RBS is where ribosomes attach to the RNA message 59 00:02:39,136 --> 00:02:42,686 so they can start linking the specified amino acids into a protein chain. 60 00:02:42,686 --> 00:02:47,526 And you can control the strength of the RBS to control how much of the message gets translated. 61 00:02:47,696 --> 00:02:48,576 >> Dude: and I'll bet there's an app for that too... 62 00:02:48,886 --> 00:02:51,836 >> Sally: well if you're interested in working on one, 63 00:02:52,076 --> 00:02:56,366 let me tell you about how genes are arranged in eukaryotic cells like plants, yeast and human... 64 00:02:56,366 --> 00:02:57,946 >> Dude: Can we do that another day Sally? 65 00:02:58,096 --> 00:02:59,996 My disk space nearly full.