WEBVTT 00:00:08.176 --> 00:00:09.106 >> Dude: Grrr! 00:00:09.306 --> 00:00:11.586 Something is really wrong with this! 00:00:11.586 --> 00:00:12.916 >> Sally: What are you playing with? 00:00:12.916 --> 00:00:15.126 >> Dude: I downloaded an app called "Part Palette." 00:00:15.126 --> 00:00:18.406 It lets me plan genetic circuits using Parts from the Registry. 00:00:18.526 --> 00:00:19.476 >> Sally: That sounds great. 00:00:19.476 --> 00:00:19.996 Can I see? 00:00:19.996 --> 00:00:22.926 >> Dude: I can select parts and then wrap them together on the palette. 00:00:22.926 --> 00:00:24.866 Or at least I'm suppose to be able to do that! 00:00:24.866 --> 00:00:24.356 I keep getting an error message. 00:00:24.406 --> 00:00:24.856 >> Sally: Hmmm... 00:00:24.976 --> 00:00:29.326 maybe the app doesn't work, or maybe there's something 00:00:29.326 --> 00:00:31.126 about the parts you're trying to wrap together. 00:00:31.436 --> 00:00:32.796 What gene are you trying to build? 00:00:32.796 --> 00:00:34.406 >> Dude: I just grabbed these 3 parts to try. 00:00:34.596 --> 00:00:38.076 >> Sally: A terminator, an operator and an open reading frame? 00:00:38.076 --> 00:00:39.366 That's not a gene... 00:00:39.366 --> 00:00:42.616 >> Dude: but it's got an open reading frame...what more do I need? 00:00:42.616 --> 00:00:43.476 >> Sally: Walk with me Dude. 00:00:43.596 --> 00:00:45.356 For starters you'll need a promoter. 00:00:45.616 --> 00:00:50.766 That's a snippet of DNA that the RNA polymerase binds to when it starts transcription. 00:00:51.146 --> 00:00:54.046 Promoters come in different strengths so choose carefully. 00:00:54.046 --> 00:00:56.446 >> Dude: How do I know if a promoter is strong or weak? 00:00:56.446 --> 00:00:59.446 >> Sally: Well the strongest promoters have this DNA sequence. 00:00:59.526 --> 00:01:02.046 RNA polymerase can bind these bases well. 00:01:02.046 --> 00:01:06.606 The more bases that are different from this consensus sequence, the weaker the promoter is 00:01:06.606 --> 00:01:11.576 since RNA polymerase will bind less well and transcribe less often...at least... 00:01:11.626 --> 00:01:13.286 that's how it usually works. 00:01:13.286 --> 00:01:13.376 >> Dude: oh no! 00:01:13.376 --> 00:01:15.386 I hear an exception coming... 00:01:15.386 --> 00:01:17.606 >> Sally: Well sure Dude...it's very clever really. 00:01:17.876 --> 00:01:20.816 There are promoters that are used in different stages of cell growth 00:01:20.816 --> 00:01:23.816 and those promoters bind a slightly different RNA polymerase 00:01:23.816 --> 00:01:26.816 so they have a consensus sequence that's a little different from this one. 00:01:26.816 --> 00:01:30.036 And then there are still other promoters that have lots of differences 00:01:30.036 --> 00:01:33.086 from this consensus sequence but they are still strong promoters 00:01:33.086 --> 00:01:37.096 since they also have DNA sequences nearby that bind activating proteins. 00:01:37.456 --> 00:01:40.186 The activators help RNA polymerase find the promoter 00:01:40.226 --> 00:01:42.246 and that gets transcription really cranking. 00:01:42.246 --> 00:01:44.366 Activated promoters can be very strong. 00:01:44.426 --> 00:01:45.866 >> Dude: When does it end!?! 00:01:45.936 --> 00:01:48.996 >> Sally: Another great question Dude!...there are sequences that come 00:01:48.996 --> 00:01:51.076 after the open reading frame called terminators. 00:01:51.186 --> 00:01:55.346 These tell RNA polymerase to stop transcribing RNA and to leave the DNA. 00:01:55.346 --> 00:01:59.956 These terminators are really useful if you have another promoter downstream of this gene. 00:02:00.296 --> 00:02:02.986 You'll need a terminator if you don't want the polymerase to read 00:02:02.986 --> 00:02:05.166 through this gene into the next one downstream. 00:02:05.236 --> 00:02:06.226 >> Dude: "Downstream?" 00:02:06.466 --> 00:02:08.515 Don't you mean "on the right?" 00:02:08.596 --> 00:02:10.826 >> Sally: Cells don't know their right from their left so we have 00:02:10.826 --> 00:02:14.566 to use the natural polarity of double stranded DNA when we give directions. 00:02:14.756 --> 00:02:18.776 "Downstream" means "more 3-prime" no matter which strand of the DNA you're working on. 00:02:18.776 --> 00:02:22.056 >> Dude: So in this app I can wrap any promoter, 00:02:22.056 --> 00:02:26.806 ORF and terminator together, and I've got a gene! 00:02:26.806 --> 00:02:28.816 >> Sally: Well actually you've got a transcription unit, 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. 00:02:32.746 --> 00:02:36.156 To translate it, you'll need to include a ribosome binding site. 00:02:36.156 --> 00:02:39.076 The RBS is where ribosomes attach to the RNA message 00:02:39.136 --> 00:02:42.686 so they can start linking the specified amino acids into a protein chain. 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. 00:02:47.696 --> 00:02:48.576 >> Dude: and I'll bet there's an app for that too... 00:02:48.886 --> 00:02:51.836 >> Sally: well if you're interested in working on one, 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... 00:02:56.366 --> 00:02:57.946 >> Dude: Can we do that another day Sally? 00:02:58.096 --> 00:02:59.996 My disk space nearly full.