| --- Day changed Tue Jun 03 2008 | ||
| joshcryer | !help | 00:42 |
|---|---|---|
| joshcryer | Denied! :( | 00:42 |
| kanzure | joshcryer: Hello. | 00:44 |
| Vedestin | krebs is not servicing the channel it seems | 00:48 |
| kanzure | do we want krebs servicing the channel? | 00:49 |
| Vedestin | did it do anything | 00:50 |
| kanzure | it told epitron secrets. | 00:51 |
| Vedestin | hmm | 00:51 |
| joshcryer | Hey kanzure | 00:52 |
| kanzure | What's up? | 00:53 |
| joshcryer | Listening to the Phoenix briefing cast got me thinking. | 00:53 |
| joshcryer | They talked about TEGA and how it seperated partciles by ionizing them and using magnetic fields to push them along. | 00:53 |
| joshcryer | (Since matter itself is non-magnetic without being ionized.) | 00:54 |
| joshcryer | (briefing cast = the mp3 of the phone briefing they had) | 00:54 |
| joshcryer | Anyway, it sounded interesting enough to discuss. | 00:54 |
| kanzure | I don't understand. The magnetic fields would push the ions along? | 00:54 |
| joshcryer | Yeah | 00:55 |
| joshcryer | They heat up some soil. | 00:55 |
| kanzure | I think the idea of the ion Hull reactors used to be that the ions would eventually just squeeze their way out or something. | 00:55 |
| joshcryer | It breaks down into the constituant compounds (it's not hot enough to make them split up into their elements). | 00:55 |
| kanzure | Oh, the actual lander. | 00:55 |
| joshcryer | And since a water particle isn't exactly magnetic they ionize it. | 00:55 |
| joshcryer | So that magnetic fields can interact and they can hold it in a nice plasma for spectromoy. | 00:56 |
| kanzure | Yes, yes, but once they have that plasma, how is this used for propulsion ? | 00:56 |
| kanzure | I mean, pushing the ions along, this would create thrust, yes? | 00:56 |
| joshcryer | We're not talking about propulsion. :P | 00:57 |
| joshcryer | We're talking about soil analysis. :) | 00:57 |
| kanzure | bleh | 00:57 |
| kanzure | Okay :) | 00:57 |
| joshcryer | I think it applies though. | 00:57 |
| joshcryer | We know the carbothermic reaction can seperate elements pretty effectively. | 00:57 |
| kanzure | So, mass spec with magnets? | 00:57 |
| joshcryer | The question is, how do you engineer something that does the chemistry? | 00:57 |
| kanzure | or at least magnets to do separation, hrm | 00:57 |
| joshcryer | That's what I was thinking. | 00:57 |
| kanzure | What's the exact carbothermic reaction? | 00:57 |
| joshcryer | Magnetic centrafuge thing. | 00:58 |
| joshcryer | It's on page 9 of that paper. | 00:58 |
| * kanzure is ashamed that he hasn't gotten to it. | 00:58 | |
| joshcryer | Pfft, it's a nice diagram. | 00:59 |
| kanzure | Actually, my time has been spent reviewing the classic: | 00:59 |
| kanzure | http://heybryan.org/~bbishop/docs/ellingtonia/2008-03-23/Construction%20of%20an%20in%20vitro%20bistable%20circuit%20from%20synthetic%20transcriptional%20switches.pdf | 00:59 |
| kanzure | It's for a group I just joined today -- first day at the lab. yay \o/ | 00:59 |
| kanzure | http://en.wikipedia.org/wiki/Carbothermic_reaction 'Carbothermic reactions are thermic chemical reactions which use carbon as the reducing agent at high temperature. The most prominent example is used in iron ore smelting.' | 01:00 |
| kanzure | hrm. | 01:00 |
| joshcryer | To me the paper itself is pretty much irrelevant. | 01:02 |
| joshcryer | It just proves what we already know. | 01:02 |
| joshcryer | http://i32.tinypic.com/14ufaes.png | 01:02 |
| joshcryer | What I think is important, at least to me, perhaps it's stupid, is the element seperation cycle expounded upon there. | 01:02 |
| kanzure | Uhh, where'd that come from? | 01:03 |
| kanzure | I mean, who acquired that information and how ? | 01:03 |
| joshcryer | It's from the paper I sent you... page 9. | 01:03 |
| kanzure | right, but | 01:03 |
| joshcryer | Lackner wrote the paper, but I'd have to look at it more closely to see where he got the chemistry. | 01:03 |
| * kanzure grumbles. Guess I really have to read it now. | 01:04 | |
| joshcryer | JANAF thermodynamic tables. | 01:04 |
| joshcryer | http://www.nist.gov/srd/jpcrd_28.htm | 01:05 |
| joshcryer | Holy shit. | 01:05 |
| joshcryer | The book is $305. | 01:05 |
| kanzure | heh' libraries at universities will have it. | 01:05 |
| joshcryer | To check out? | 01:06 |
| Vedestin | shortloans atleast | 01:07 |
| joshcryer | Probably not for non-students though, I imagine? | 01:07 |
| joshcryer | (Obviously I'd want to scan it in. ;)) | 01:07 |
| Vedestin | not to check out, now | 01:07 |
| Vedestin | they have scanners in uni libraries | 01:07 |
| joshcryer | Barns and Noble has it for $195. | 01:07 |
| joshcryer | I'll see if the local universities have it. | 01:07 |
| Vedestin | yeah, university has it for free | 01:08 |
| kanzure | joshcryer: I've been able to walk into uni libraries quite easily. | 01:08 |
| kanzure | portable scanner :) | 01:08 |
| joshcryer | kanzure, same here. | 01:08 |
| Vedestin | anyone can walk in to a uni library | 01:09 |
| joshcryer | All schools here are open at least. | 01:09 |
| joshcryer | Vedestin, well some schools are crap and locked down because of the shootings. | 01:09 |
| Vedestin | isnt that just high schools | 01:09 |
| Vedestin | that's barely a uni at all | 01:09 |
| joshcryer | V. Tech is all locked down now. | 01:09 |
| Vedestin | who the fuck would lockdown | 01:09 |
| kanzure | uh | 01:10 |
| kanzure | virginia tech got slaughtered | 01:10 |
| joshcryer | Yeah | 01:10 |
| Vedestin | i remember that | 01:10 |
| Vedestin | cho | 01:10 |
| joshcryer | Worst in American history, so it's no surprise they locked dow. | 01:11 |
| Vedestin | cho was a student | 01:11 |
| Vedestin | he'd have gotten in anyway | 01:11 |
| joshcryer | But yeah when I had no computer I would walk into uni library and just use internet. | 01:11 |
| joshcryer | They don't bug you unless it's exams time. | 01:11 |
| joshcryer | Then they'll ask if you're a student. | 01:11 |
| Vedestin | are you homeless? | 01:11 |
| joshcryer | LOL, no. | 01:11 |
| Vedestin | why would they bug you at all then | 01:12 |
| joshcryer | I went on, a waltzing matilda before though. | 01:12 |
| Vedestin | i see | 01:12 |
| joshcryer | Because if it's exams time and you look like a non-student and computers are needed they'll ask you for your student ID. | 01:12 |
| Vedestin | libraries are always full of mature-age students | 01:12 |
| Vedestin | because kids have their own computers | 01:13 |
| kanzure | uh? | 01:13 |
| joshcryer | Yeah | 01:13 |
| joshcryer | Now adays it doesn't really matter. | 01:13 |
| joshcryer | Students use their own computers. | 01:13 |
| kanzure | Have you been to a public high school library ? | 01:13 |
| joshcryer | But back in 2002 I went to a university and it was exams time and they booted me. | 01:13 |
| Vedestin | public high school isn't university | 01:14 |
| joshcryer | It was in Clearwater OK. | 01:14 |
| joshcryer | Can't remember the university. | 01:14 |
| Vedestin | lol | 01:14 |
| joshcryer | But w/e, it's a moot point. | 01:14 |
| joshcryer | I'll see if The University of Colorado has a copy. | 01:14 |
| joshcryer | Let's see if I can get on their website. | 01:15 |
| joshcryer | Barns & Nobel has it for $160. | 01:15 |
| Vedestin | buy it if you can afford it | 01:15 |
| joshcryer | I think I might. | 01:16 |
| Vedestin | i just failed first semester | 01:17 |
| joshcryer | Aww. | 01:17 |
| joshcryer | What were you taking? | 01:17 |
| Vedestin | math 111, chem 101, engineering 100 and phys 120 | 01:18 |
| Vedestin | i withdrew from phys straight up | 01:18 |
| Vedestin | then i withdrew from eng | 01:18 |
| joshcryer | You exploded bro. | 01:18 |
| Vedestin | now i realise i have my final exam for math next week and i haven't been to a lecture in 3 months | 01:18 |
| joshcryer | Gotta take it easy. | 01:18 |
| joshcryer | That's some hard stuff. | 01:18 |
| Vedestin | four courses per semester is the norm | 01:18 |
| Vedestin | plus, i've been out of high school for a few years, so i couldn't remember anything | 01:19 |
| Vedestin | only thing i'll pass is chem | 01:19 |
| joshcryer | Library use only. | 01:20 |
| joshcryer | That's a shame. | 01:20 |
| Vedestin | there are scanners in the library | 01:20 |
| Vedestin | flatbeds atleast | 01:20 |
| Vedestin | or there should be | 01:20 |
| joshcryer | The 1986 version is for checkout though. | 01:20 |
| joshcryer | I imagine that one is good enough. | 01:20 |
| joshcryer | Who needs the 1998 version. | 01:20 |
| Vedestin | get that then | 01:21 |
| joshcryer | I need to find someone enrolled... ;) | 01:22 |
| Vedestin | oh yeah, right | 01:25 |
| Vedestin | you could just steal a wallet | 01:26 |
| joshcryer | Nah. | 01:26 |
| Vedestin | i bet it's a self checkout service | 01:26 |
| joshcryer | I'm sure someone would be helpful. | 01:26 |
| Vedestin | really? | 01:26 |
| joshcryer | In fact my brother is enrolling there this summer. | 01:26 |
| Vedestin | there's no way i'd let some random check a book out on my record | 01:26 |
| joshcryer | I could just wait. | 01:26 |
| joshcryer | Or buy it at B&N. | 01:26 |
| joshcryer | Vedestin, even if I offered you dinner and let you watch me scan the tables in? | 01:26 |
| joshcryer | (Not the whole book, just the important parts.) | 01:27 |
| Vedestin | would you cook yourself? | 01:27 |
| joshcryer | Haha | 01:27 |
| joshcryer | Why yes, yes I would. | 01:27 |
| joshcryer | What would you like to eat? | 01:27 |
| Vedestin | nah, i don't want you to cook | 01:27 |
| Vedestin | we'll just get chinese | 01:27 |
| Vedestin | seriously though, there'd be scanners at the library | 01:27 |
| joshcryer | Yeah I know. | 01:28 |
| joshcryer | I'll go check it out. | 01:28 |
| Vedestin | k bye | 01:28 |
| joshcryer | Haha | 01:28 |
| joshcryer | It's 11:30PM here. | 01:28 |
| Vedestin | uni libraries are 24 hour | 01:28 |
| joshcryer | Yeah but. | 01:29 |
| joshcryer | I think I'd be more obvious going in at night. | 01:29 |
| Vedestin | maybe they have a security dude or something | 01:33 |
| Vedestin | couldnt you just get a library card from them | 01:34 |
| joshcryer | Haha | 01:34 |
| Vedestin | not from the security guard | 01:35 |
| Vedestin | from the university | 01:35 |
| Vedestin | enrol in a non-award program | 01:35 |
| Vedestin | get a student card | 01:35 |
| Vedestin | and you're done | 01:35 |
| Vedestin | free books | 01:35 |
| Vedestin | forevers | 01:35 |
| joshcryer | http://www.nist.gov/srd/PDFfiles/jpcrdM9.pdf | 02:07 |
| joshcryer | It's online! | 02:07 |
| joshcryer | How did I overlook that? | 02:07 |
| joshcryer | 250mb pdf file lol | 02:07 |
| Vedestin | hahaha | 02:18 |
| joshcryer | if this was OCR'd it'd be 25 mb | 02:20 |
| ybit | http://books.google.com/books?id=8axpjcE0bqUC&dq=%E2%80%9CBrain-Implantable+Biomimetic+Electronics+as+a+Neural+Prosthesis+for+Hippocampal+Memory+Function,%E2%80%9D&source=gbs_summary_s&cad=0 | 02:21 |
| ybit | 1:22am here | 02:22 |
| ybit | thought i'd chime in :) | 02:22 |
| joshcryer | This book has every single chemical composition known to man. | 02:49 |
| joshcryer | And formulas how to get to each one. | 02:49 |
| joshcryer | This is all you need! | 02:49 |
| joshcryer | Any other channels like this that aren't so dead? | 03:59 |
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| faceface | can you give me an implant to speak german? | 07:32 |
| nsh | the you that speaks german is discontinuous from the you that doesn't | 07:36 |
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| -!- Topic for #hplusroadmap: http://heybryan.org/ http://heybryan.org/mediawiki/ http://heybryan.org/exp.html | krebs is now servicing the channel. try !help | 07:44 | |
| -!- Topic set by kanzure [] [Tue Apr 29 18:54:31 2008] | 07:44 | |
| [Users #hplusroadmap] | 07:44 | |
| [ faceface] [ fenn_ ] [ nsh ] [ Vedestin] | 07:44 | |
| [ fenn ] [ kanzure] [ Phreedom] [ ybit ] | 07:44 | |
| -!- Irssi: #hplusroadmap: Total of 8 nicks [0 ops, 0 halfops, 0 voices, 8 normal] | 07:44 | |
| -!- [freenode-info] if you need to send private messages, please register: http://freenode.net/faq.shtml#privmsg | 07:44 | |
| -!- Channel #hplusroadmap created Sat Mar 22 15:44:12 2008 | 07:45 | |
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| kanzure | faceface: Hey. | 08:13 |
| faceface | yo k | 08:20 |
| faceface | I figure, what would I really want an implant for? | 08:20 |
| faceface | I would like a dictionary of german words and phrases | 08:20 |
| faceface | how could we hook up such a memory expansion? | 08:21 |
| faceface | would that 'brain interferance' technology that got posted a while back (the one that stopped a guy from talking using a targeted magnetic field) be used in a refined way to 'implant' certain thoughts? | 08:21 |
| faceface | let me put my thinking cap on... | 08:22 |
| Vedestin | it wouldn't | 08:22 |
| faceface | Vedestin: wouldn't what? | 08:22 |
| Vedestin | it's like how you use the great wall of china to do brain surgery | 08:22 |
| faceface | Vedestin: sure, at current resolutions | 08:22 |
| faceface | Won't the resolution get driven down by better magnets (super conductors) and better software | 08:23 |
| faceface | ? | 08:23 |
| Vedestin | probably not | 08:23 |
| Vedestin | not to a level that you'd need atleast | 08:23 |
| faceface | MRI -> MFRNI | 08:23 |
| faceface | Vedestin: but what 'level' would be interesting? currently you can target the 'speach centers', surly we can do more intereseting thigs with higher res | 08:24 |
| faceface | what about focusing attention on the 'forign language' centers of the brain? (I heard that adults who become bilingual use a different region of their brain to talk forign than they do to talk their mother tongue) | 08:25 |
| faceface | what about mildly surpressing the 'mother tongue' brain function while mildly stimulating the forign language centers during an intensive dictionary crash course? | 08:26 |
| faceface | of course it could backfire and leave you talking less of the forign language than controll | 08:26 |
| faceface | Vedestin: my point is that refining the 'level' will always be 'interesting', therefore high levels of resolution should be acheived naturally | 08:26 |
| faceface | by continuious improvement driven by better technology | 08:27 |
| faceface | anyway... let me know when we can start de deutsche sprachen. | 08:27 |
| -!- You're now known as fenn | 08:32 | |
| Vedestin | faceface, it really doesn't sound like the way to go | 08:32 |
| kanzure | http://en.wikipedia.org/wiki/Friendly_artificial_intelligence <-- I see. Eli got the nod from a few 'professionals'. | 08:36 |
| kanzure | faceface: http://heybryan.org/mediawiki/index.php/rTMS | 08:36 |
| kanzure | faceface: Your idea of supressing the mother tongue is interesting. | 08:36 |
| * nsh is dubious | 08:37 | |
| kanzure | nsh: It would be fun to try. | 08:37 |
| nsh | just wondering about children who are raised bilingually | 08:37 |
| nsh | also, don't you mean: it'd be fun to try on a neural slice or something else that makes your earlier disparagement of chemical neuromodification experiments non-hypocritical? :-) | 08:38 |
| kanzure | http://www.singinst.org/ourresearch/publications/what-is-friendly-ai.html | 08:43 |
| kanzure | nsh: No, I really mean it'd be fun to use rTMS :) | 08:43 |
| fenn | faceface: the problem with rTMS is it's like using a stick of dynamite, when what you really want is a chisel | 08:45 |
| fenn | and there's no obvious way to make the effect more fine grained | 08:45 |
| kanzure | I want a proof about this 'friendly ai' BS. I think the unstated fear is "well, Vinge said they will be able to catch us no matter where we go, so we're doomed!" | 09:00 |
| kanzure | doomed | 09:01 |
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| faceface | fenn: how was it 'focused' in the first place? | 10:36 |
| faceface | nsh: bilingual individuals use the same region of their brain for both languages | 10:36 |
| faceface | (unlike ppl. who learn a language in adulthood) | 10:36 |
| faceface | perhaps we could just develope a 'learn a language pill' that lets you access the 'core' language region of your brain again... or perhaps that would just result in turning you into a babbeling child | 10:37 |
| nsh | so.. beyond which stage of cognitive development does a second language require an alternate localisation? | 10:51 |
| nsh | say if you were raised bilingually until the age of 7, then spoke just one language until adolescence, then relearnt the second language... | 10:52 |
| nsh | (as is my case) | 10:52 |
| * nsh remains dubious | 10:52 | |
| faceface | nsh: interesting example | 10:57 |
| faceface | you would need to be probed ;-) | 10:57 |
| faceface | I only remember the factoid 'ppl. use a different region of their brain to talk a second language if learnt after childhood' | 10:58 |
| faceface | I could guess 7 years... but its a guess | 10:58 |
| faceface | in your case... I'd say that the bilingual impression for 7 years would not fade | 10:59 |
| faceface | you went back to your core region... I would guess.... now if you learned a 3rd language... oh my! | 10:59 |
| nsh | thing is that these summarisations of research are generally more misleading than revealing | 11:01 |
| faceface | how dare you! my left brain is insulted! | 11:01 |
| faceface | my right brain is admiring my desktop wallpaper | 11:02 |
| faceface | http://www.wellcome.ac.uk/News/News-archive/Browse-by-date/2007/News/WTD026366.htm ?? | 11:02 |
| * nsh smiles | 11:02 | |
| faceface | It has been shown that monolingual children lose at least some of their sensitivity to foreign phonemes between the age of 6 and 12 months (Werker and Tees 1984Go; Cheour and others 1998Go; Rivera-Gaxiola and others 2005Go). ??? | 11:06 |
| faceface | Pallier and others (1997)Go have shown that even early exposure to a second language before the age of 6 years is not enough to attain first language–type phonological competence. | 11:06 |
| faceface | http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WC0-49JHHF5-6&_user=28761&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000002718&_version=1&_urlVersion=0&_userid=28761&md5=7ea6388b87abe31b66062c2cf6fe7c57 | 11:08 |
| faceface | that last one is as close as I have found | 11:08 |
| --- Log opened Tue Jun 03 11:22:22 2008 | ||
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| -!- Topic set by kanzure [] [Tue Apr 29 18:54:31 2008] | 11:23 | |
| [Users #hplusroadmap] | 11:23 | |
| [ faceface] [ fenn] [ nsh] [ Phreedom] [ ybit] | 11:23 | |
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| faceface | fenn: use less dynamyte | 11:29 |
| faceface | fenn: or build something else | 11:29 |
| fenn | the nice thing about rTMS is you dont have to drill any holes in peoples' skulls | 11:39 |
| fenn | i bet there's some way to use the bessel function to recreate a desired 3D electric field in arbitrary space (but only for a short amount of time, which might still work) | 11:41 |
| fenn | or whatever the 3d version of the bessel function is called | 11:42 |
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| fenn | this is what i'm remembering re: bessel function: http://www.pinktentacle.com/2006/07/device-uses-waves-to-print-on-water-surface/ | 11:52 |
| nsh | that's cool | 11:52 |
| nsh | could be cooler | 11:53 |
| fenn | aww the video's down wtf | 11:54 |
| fenn | youtube gets suckier every day | 11:54 |
| nsh | if i was allowed anywhere near google | 11:54 |
| nsh | removed videos would automatically redirect to replacements | 11:55 |
| nsh | then again, i would probably not have videos go down, ever | 11:56 |
| nsh | but i think people would dislike that even more | 11:56 |
| fenn | people with lawyers. i think regular people wouldn't care | 11:57 |
| fenn | that think is sorta like a phased array radar | 11:58 |
| nsh | hm | 12:00 |
| nsh | bessel functions also arise in quantum mechanics | 12:03 |
| nsh | http://quantummechanics.ucsd.edu/ph130a/130_notes/node225.html | 12:03 |
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| kanzure | Hi all. | 18:07 |
| fenn | g'day | 18:07 |
| kanzure | http://grailsearch.org/?q=node/77#comment-226 <-- I just posted to grailsearch.org | 18:39 |
| kanzure | fenn: http://heybryan.org/mediawiki/index.php/Wittig_cohort but probably stuff you've already seen | 18:40 |
| kanzure | I'm pretty much very, very sure now that the majority of good work that happens, happens on your own | 18:40 |
| kanzure | since the majority of the work that I got done in the day was in a 5 minute period. | 18:41 |
| fenn | 5 minutes in the lab? | 18:42 |
| kanzure | well, on a computer | 18:43 |
| kanzure | I'm not doing anything wet since they want to do some "Don't Die" safety training courses | 18:44 |
| fenn | oh, dangerous stuff, that DNA | 18:44 |
| kanzure | which is totally BS, and they know it, they're actually waiting for a student to come back in who knows his techniques/protocols well | 18:44 |
| kanzure | right, we're not even doing radiotagging | 18:44 |
| kanzure | bbl | 18:46 |
| kanzure | okay, back | 18:52 |
| kanzure | fenn: I'm thinking of illustrating skdb with wet ware first, even though wetware has this tendency to suck | 18:52 |
| kanzure | and generally not be solid state | 18:52 |
| kanzure | so the 'bioreactor'/biotank thing | 18:52 |
| fenn | looks like a pain in the butt | 18:53 |
| fenn | oops | 18:53 |
| kanzure | yes | 18:53 |
| kanzure | but | 18:53 |
| fenn | wrong chan sry | 18:53 |
| kanzure | it'll have immediate results in that it'll be mostly self-replicating | 18:53 |
| kanzure | except for the metal tank | 18:53 |
| kanzure | and uh, maybe some other things like any electronics (which I want to keep to a minimum -- have it all automated via biology) | 18:53 |
| kanzure | actually, the tank may not be metal | 18:54 |
| kanzure | but you get the idea | 18:54 |
| fenn | hmm.. automated via biology? | 18:55 |
| kanzure | so, the baseline system will just produce the chemicals like T7 and so on, that would be necessary for transcription and doing other DNA experiments, gene splicing, insertion, plasmids, whatever | 18:55 |
| kanzure | you were mentioning in vitro synthesis via laser light flashing a few months ago -- I think I've figured out some possible implementations | 18:56 |
| kanzure | the next step up from baseline would do something, I don't know what. Personally I want to try inserting the neurotransmitter synthesis pathways into ecoli. | 18:56 |
| fenn | kainic acid stuff? i think if it were easy someone would have done it already, but maybe its just cross-discipline taboo stuff | 18:57 |
| kanzure | right, I'm not completely certain it's possible, I bet there's a lot of funky missing pieces | 18:57 |
| kanzure | the purification proc migth be it | 18:57 |
| kanzure | fenn: looks like I might have a contract with somebody to extend drupal.org software | 19:13 |
| kanzure | hurray for PHP? | 19:14 |
| fenn | a contract? | 19:14 |
| kanzure | well, I'm waiting for him to email it to me | 19:14 |
| kanzure | but I just got off the phone and it sounds good | 19:14 |
| kanzure | uhm, so, the purification stuff is what I'm worrying about | 19:16 |
| kanzure | affinity chromatography sounds like it takes some pretty funky equipment | 19:16 |
| fenn | its not just a column with various solutions passing through it? | 19:18 |
| kanzure | ok, please explain column chromatography setups | 19:18 |
| kanzure | I've read wikipedia, I've read other docs, it just doesn't make sense | 19:18 |
| kanzure | paper chromatography, being the first type I learned about, has completely thrown me off base | 19:18 |
| fenn | so there's a column, meaning a cylindrical plug of goo in a syringe or a glass tube | 19:19 |
| fenn | you can add water to the column and it slowly oozes through the plug of goo | 19:20 |
| fenn | the plug is the 'substrate' which has an affinity for what you're trying to purify | 19:20 |
| kanzure | goo could equal wha? something like, a gel? | 19:20 |
| fenn | so, you'd bind your DNA to that | 19:20 |
| kanzure | hrm | 19:20 |
| fenn | yeah it looks like agarose (at least when i did it) | 19:20 |
| kanzure | this is column? | 19:21 |
| fenn | yes | 19:21 |
| kanzure | okay, so how do we come up with affinities for specific molecules? | 19:21 |
| kanzure | computational chem? | 19:21 |
| fenn | natural selection? | 19:21 |
| fenn | aptamer process | 19:21 |
| fenn | mm.. i guess you have to have a sample of what you want before you can do that | 19:22 |
| kanzure | aptamer process sucks apparently, I've asked the Ellington goons | 19:22 |
| kanzure | I mean, for purification | 19:22 |
| fenn | why? | 19:22 |
| kanzure | for selection, like bonding to receptors, it works | 19:22 |
| kanzure | same reason you mentioned | 19:23 |
| fenn | not specific enough? | 19:23 |
| kanzure | 'sample of what you want before in the first place' | 19:23 |
| kanzure | specificity has to increase with bp length :( | 19:23 |
| kanzure | so 100 bp might not be enough in some cases, apparently | 19:23 |
| fenn | well once you have the aptamer then you sequence it and anyone can use it | 19:23 |
| kanzure | and as you increase bp, the DNA synthesizers quickly begin to suck | 19:23 |
| kanzure | (in vitro DNA synthesis, again, might be useful) | 19:23 |
| kanzure | right, or just copy them | 19:23 |
| kanzure | aptamer -> denature -> clone -> ... | 19:24 |
| fenn | can use a western blot to purify small samples of protein | 19:24 |
| fenn | works sorta like 2 dimensional paper chromatography | 19:24 |
| fenn | you end up with a piece of paper with dots all over it | 19:24 |
| kanzure | I've heard about blots, but I still have't internalized | 19:24 |
| kanzure | oh, blot == just a gel? | 19:24 |
| fenn | its filter paper | 19:24 |
| fenn | but works sorta like a gel | 19:24 |
| kanzure | so the filter is specific to what you're blotting? | 19:25 |
| fenn | oh sorry the actual separation is performed in a gel | 19:26 |
| kanzure | oh | 19:26 |
| fenn | and then it gets 'blotted' onto filter paper | 19:26 |
| kanzure | wait, I misinterpreted your recommendation | 19:26 |
| kanzure | "natural selection? aptamer process" | 19:26 |
| kanzure | I didn't read the implied "i.e., aptamer process" | 19:26 |
| kanzure | the implied "i.e." | 19:26 |
| kanzure | hrm, the same comments we mentioned still apply :) nevermind | 19:26 |
| kanzure | so, bruteforcing purification methodologies: physical/mechanical, chem, electrical, thermo, nuclear (radioactivity & decay), surface tension (according to Wikipedia article on energy) | 19:29 |
| fenn | since its a recombinant protein, you could 'tag' the protein with a covalently linked 'handle' of a well-characterized binding complex | 19:29 |
| kanzure | physical - crank up the RPMs via centrifuges, phase changes (affinity chromatography), | 19:30 |
| kanzure | well, it's multiple proteins of course | 19:30 |
| fenn | and then just cleave the handle off with a digestion enzyme | 19:30 |
| fenn | you could use the same method each time | 19:30 |
| kanzure | oh | 19:30 |
| kanzure | what if the handle is degradable? | 19:30 |
| kanzure | that would be useful | 19:30 |
| kanzure | once you have it purified, you degrade the handle, inject into human | 19:30 |
| kanzure | yeah, cleave | 19:30 |
| kanzure | so, there's a known binding complex that we can use? | 19:31 |
| fenn | Adding a tag to the protein such as RuBPS gives the protein a binding affinity it would not otherwise have. Usually the recombinant protein is the only protein in the mixture with this affinity, which aids in separation. The most common tag is the Histidine-tag (His-tag), that has affinity towards nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions on a resin, an affinity support that specifically binds to histidine-tagged proteins can be created. | 19:31 |
| kanzure | cutoff@histidin (support that specifically binds to histidin) | 19:32 |
| fenn | right | 19:32 |
| kanzure | but I get it | 19:32 |
| fenn | there is probably a more biological version that works similarly | 19:32 |
| kanzure | let's say that we have a tank of cells (or even just a tank of ribo* stuff, but I think we need cells still) that have transcribed & translated & made the protein we want, with the RuBPS | 19:32 |
| kanzure | so, how would we do this? would we cycle water over the culture, | 19:33 |
| kanzure | or would we centrifuge the hell out of it? | 19:33 |
| fenn | i like those beads actually | 19:33 |
| kanzure | and then take the remaining supernatant / goo and run it through something that has surface bonds of | 19:33 |
| kanzure | yeah, so beads on a surface? | 19:33 |
| kanzure | actually, I suggested earlier today to a friend to use nanoparticles in the blood stream | 19:33 |
| kanzure | for something totally different | 19:33 |
| fenn | beads in solution, centrifuge, decant, elute beads | 19:33 |
| kanzure | if the nanoparticles could be used to bind to the RuBPS handles | 19:33 |
| kanzure | in context, we were discussing myostatin and muscle growth in marathon mice and hu | 19:34 |
| kanzure | the poor grad student (Mike Wittig) has realized that I'm pretty well read | 19:34 |
| fenn | heh | 19:34 |
| kanzure | fun guy, Mike. | 19:34 |
| kanzure | btw, Mike has a friend at UT somewhere, some Russian guy named Stan, that does multimonitor setups. he also did an IP-by-IP scan of the women's dorms to figure out which rooms had linux installations ... | 19:35 |
| kanzure | heh' | 19:35 |
| kanzure | IP blocks rock in that way. | 19:36 |
| fenn | dunno what to think about that | 19:36 |
| kanzure | fenn: don't you and I googlestalk anyway? | 19:36 |
| fenn | i plead the fifth | 19:36 |
| kanzure | anyway | 19:36 |
| kanzure | how do you get the molecules off of the beads | 19:36 |
| fenn | cleave the tag | 19:37 |
| kanzure | the tag is in between the molecule and the surface | 19:37 |
| fenn | or some salty magick | 19:37 |
| kanzure | not physically accessible | 19:37 |
| fenn | i was thinking there's a long chain between the two proteins | 19:37 |
| kanzure | hrm, salty magik -> degrade the proteins, they detach, but then reform when solution returns to normal ? | 19:37 |
| kanzure | perhaps | 19:37 |
| kanzure | I'm not sure, how would we make sure? is that common? | 19:37 |
| fenn | make sure what? | 19:37 |
| kanzure | that there's a good space between the protein and the nanoparticle | 19:37 |
| kanzure | hrm, buckyballs | 19:37 |
| fenn | uh.. you write it into the sequence | 19:38 |
| kanzure | huh? | 19:38 |
| kanzure | the protein can just randomly be mutated like that? | 19:38 |
| fenn | gene start... protein A... chain ... protein B ... end | 19:38 |
| kanzure | to be given such a 'tail' ? | 19:38 |
| kanzure | cool | 19:38 |
| kanzure | I think that's something involving hydrogen bonds | 19:38 |
| kanzure | I remember something about specific protein bonding mechanisms | 19:38 |
| fenn | you could build it up that way, but it's a lot more work | 19:39 |
| kanzure | the beads are ridiculously easy to synthesize | 19:42 |
| kanzure | now, the surface chem to bind the RuBPS-gropers to the nanoparticle? | 19:43 |
| kanzure | actually, it doesn't need to be nano | 19:43 |
| kanzure | but nano maximizes surface area | 19:43 |
| kanzure | http://postbiota.org/pipermail/tt/2008-February/002547.html only thing wrong with this page - Gold is not a semiconductor. | 19:45 |
| kanzure | I quickly corrected myself: http://postbiota.org/pipermail/tt/2008-March/002548.html | 19:45 |
| kanzure | so, fenn, remember how you were talking about in vivo sequencing/synthesis a while back? | 19:52 |
| Splicer | i know very little about protein purification... i just started reading about it a few days ago... my image of it was that the standard method was affinity binding with receptor coated plastic beans in columns.. and that the receptors were synthezized to match the protein one wanted bound... am I totally off? Or is there some reason the 'add standard tag - catch the proteins with it - cleave the tag off' procedure is not used? | 19:52 |
| kanzure | fenn: what we're building in the lab is a way to translate frequency (like flashing photon input) into kinetical changes; | 19:52 |
| kanzure | perhaps this could be a way to control transcription? | 19:53 |
| kanzure | I don't actually recall the exact method that you were suggesting | 19:53 |
| fenn | sure, hamad schifferle (?) at CBA was doing gold nanoparticles that caused DNA to unravel when exposed to radio | 19:56 |
| fenn | god CBA's website really sucks | 19:56 |
| fenn | unless that was some weird dream i'm remembering | 19:57 |
| kanzure | no, CBA really sucks | 19:57 |
| kanzure | (web) | 19:57 |
| fenn | Remote Electronic Control of DNA Hybridization Through Inductive Coupling to an Attached Metal Nanocrystal Antenna, K. HamadSchifferli, | 19:58 |
| fenn | ah its one of those hybrid frankenstein names | 19:59 |
| kanzure | okay, that's close | 20:00 |
| fenn | http://web.mit.edu/be/people/hamad.htm | 20:00 |
| kanzure | but that's requiring metal | 20:00 |
| kanzure | we're building a biological equivalent | 20:00 |
| fenn | yeah should be able to do similar with quinones, fluorescins, rhodopsin, chlorophores | 20:00 |
| fenn | basically anything colorful | 20:00 |
| fenn | maybe could hack some existing light-activated enzymes | 20:01 |
| kanzure | nono, | 20:02 |
| kanzure | we have a represillator/oscillator -- a circuit of transcriptional switches going around in a circle | 20:02 |
| kanzure | light-activated enzymes would be useful, I suppose | 20:02 |
| fenn | i dont know what 'represillator' means | 20:02 |
| kanzure | but it's not in the transcriptional logic itself | 20:02 |
| kanzure | represillator == oscillator | 20:02 |
| kanzure | represillator = repressor/activator oscillator | 20:02 |
| fenn | ok | 20:02 |
| fenn | stupid name | 20:02 |
| fenn | ;) | 20:02 |
| kanzure | heh :) | 20:02 |
| fenn | so, that's your clock cycle? | 20:03 |
| kanzure | yes | 20:03 |
| kanzure | fenn: I could send you a few papers | 20:03 |
| fenn | then you only need one 'data line' for asynchronous serial | 20:04 |
| kanzure | I'll have to show you the paper, yeah | 20:04 |
| fenn | i think the hard part is getting it all to work :) | 20:04 |
| kanzure | sure | 20:05 |
| kanzure | we're doing just one section of the larger schematic at the top of the page on, uh, page 7 | 20:05 |
| kanzure | but I'm dubious about the memory state system -- I think it can work, but it's hard to get people understanding that it's not quite like physical RAM address space | 20:05 |
| kanzure | hrm, anyway | 20:07 |
| fenn | what you trying to do overall? | 20:07 |
| kanzure | we're making the repressilator component in the diagram on pg 7 | 20:08 |
| kanzure | that's in the lab | 20:08 |
| kanzure | now, what I want to do re: your "shine a light to synthesize DNA" system | 20:08 |
| kanzure | the lab project allows that | 20:08 |
| fenn | oh, i thought you were just pulling a repressilator off the shelf and using it for someting else | 20:08 |
| kanzure | nah | 20:08 |
| kanzure | as I was saying - the light frequency input - can be done via this method. So if you can program the state machine with pulsing photons, then the synthesizing component needs to come into play to do it nucleotide-by-nucleotide with the following requirements: | 20:09 |
| kanzure | (1) not interfering with the existing template/non-template switch and source strands | 20:10 |
| kanzure | (2) lots of nucleotides flying around ready to be used to make the next piece of the DNA strand | 20:10 |
| kanzure | (3) looks like 2 bits of memory? since we need 4 different nucleotide possibilities? | 20:10 |
| kanzure | but then we need some more data, actually | 20:10 |
| kanzure | since there's also stuff like how to end the molecule | 20:10 |
| kanzure | (it'll just be controlling the oligonucleotide reaction process) | 20:10 |
| kanzure | really, I'm hoping that oligo synthesis could work with these transcriptional elements nearby, otherwise the idea's foobarred | 20:11 |
| fenn | wtf are you talking about memory? | 20:11 |
| fenn | and state machines? | 20:11 |
| kanzure | did you look at the diagram? | 20:12 |
| fenn | no, where is that? | 20:12 |
| kanzure | email, in the pdf page 7 | 20:12 |
| kanzure | top diagram | 20:12 |
| kanzure | I'm not sure if we actually need 'memory' for this | 20:13 |
| kanzure | if we need more than 1 bit, then yes | 20:13 |
| kanzure | since to do an adenosine versus guanine you'd have to do 2 pulses, etc. | 20:13 |
| kanzure | 4 nucleotides, I mean | 20:14 |
| kanzure | so that's at least 2 bits | 20:14 |
| kanzure | and then there's a few other details that might have to be implemented ... like the way to end the strand or something? I'm not entirely versed in oligo synthesis parameters. | 20:14 |
| kanzure | or the variations to the protocol, I mean. | 20:14 |
| fenn | yep 4 nucleotides needs 2 bits, but i'd settle for 2 nucleotides | 20:15 |
| kanzure | how so? | 20:16 |
| fenn | uh, nevermind | 20:16 |
| kanzure | in terms of awesomeness, or a way to | 20:16 |
| kanzure | yeah | 20:16 |
| fenn | i dont think there exist any enzymes to synthesize a dna or rna strand from nothing at all | 20:16 |
| fenn | i'm thinking something involving tRNA.. not sure why | 20:17 |
| kanzure | wait, this is going to suck | 20:19 |
| kanzure | transcriptional switches take forever | 20:19 |
| fenn | yes | 20:19 |
| kanzure | but it might be faster than building a DNA synthesizer | 20:19 |
| kanzure | would it? | 20:19 |
| fenn | if you have a dna lab to work in :0 | 20:20 |
| kanzure | oh | 20:20 |
| kanzure | re: 2 nucleotides | 20:20 |
| fenn | uh, if you get it working it would be worth zillions | 20:20 |
| kanzure | just have two different colonies | 20:20 |
| kanzure | each with 2 bit input | 20:20 |
| kanzure | i.e., light/dark maybe | 20:20 |
| kanzure | and then integrate or something | 20:20 |
| kanzure | hrm | 20:20 |
| kanzure | that wouldn't work | 20:20 |
| kanzure | (zipper strategy) | 20:21 |
| fenn | i was thinking 3 wavelengths of light | 20:21 |
| kanzure | one colony does A & T, the other does G & C. but the zipper issue ... lineup, etc ... | 20:21 |
| kanzure | well, light/dark is easy because you can just cover it up or not | 20:21 |
| fenn | but that doesnt fit the ellingtonia agenda | 20:21 |
| fenn | need to squeeze transcriptional switches into the design somewhere :) | 20:21 |
| kanzure | heh | 20:21 |
| fenn | could build a demultiplexer out of trans switches | 20:21 |
| kanzure | basically I'm ok with needing multiple light sources, but it needs to be something doable on the cheap | 20:22 |
| kanzure | for example, building fires that burn different colors | 20:22 |
| kanzure | like distinct colors. | 20:22 |
| fenn | uhh whats wrong with an LED? | 20:22 |
| kanzure | can you make an LED? | 20:22 |
| fenn | no | 20:22 |
| kanzure | can you bash rocks together to make sparks? | 20:22 |
| fenn | ok i see where you're going | 20:22 |
| kanzure | :) | 20:22 |
| fenn | hmm. what kind of tech level is allowed? | 20:23 |
| kanzure | that's a good question | 20:23 |
| kanzure | none would be awesome | 20:23 |
| kanzure | but I'm willing to compromise | 20:23 |
| fenn | fluorescent tubes give off different kinds of light | 20:23 |
| fenn | depending on what gas is in it | 20:23 |
| kanzure | the way that I seem to be working here is starting with 'advanced tech' and then finding a simplification of it | 20:23 |
| kanzure | vacuum tubes? | 20:23 |
| fenn | geissler tubes | 20:23 |
| kanzure | we talked about these before methinks | 20:24 |
| kanzure | glassblowing sucks | 20:24 |
| kanzure | or blows | 20:24 |
| fenn | just a glass tube filled with gas and HV | 20:24 |
| fenn | anyway it's 1800's tech | 20:24 |
| kanzure | but if necessary, glassblowing is acceptable | 20:24 |
| fenn | i'm trying to think of a way to convert electrical signals to light signals using biological materials | 20:24 |
| kanzure | oh, so different gases burn different colors, right? | 20:24 |
| kanzure | uhh | 20:24 |
| fenn | like frankenstein firefly | 20:25 |
| fenn | bzzt bztzt | 20:25 |
| kanzure | so, organic LEDs were in the news recently | 20:25 |
| kanzure | fluoroproteins | 20:25 |
| kanzure | now we need a bioluminescent, electrical protein | 20:25 |
| kanzure | what do fireflies use anyway? | 20:25 |
| fenn | luciferase | 20:25 |
| kanzure | enzyme reaction? | 20:25 |
| kanzure | 'Light production in fireflies is due to a chemical reaction that occurs in specialized light-emitting organs, usually on the lower abdomen. The enzyme luciferase acts on luciferin in this organ to stimulate light emission.' | 20:26 |
| kanzure | surely different fireflies do different colors? | 20:26 |
| fenn | its activated by oxygen, the blinking is due to their flying up/down cycle which increases respiration | 20:26 |
| kanzure | hell, let's do a selection experiment on firefly light color | 20:26 |
| kanzure | wouldn't be too hard to set up a firefly cage | 20:26 |
| fenn | i've only seen green fireflies | 20:26 |
| kanzure | surely it's mutatable? | 20:26 |
| kanzure | aha | 20:26 |
| kanzure | ellingtonia specializes in knocking out nucleotides in genes | 20:27 |
| fenn | i know there are blue and red light-emitting proteins in deep sea creatures | 20:27 |
| kanzure | with unnatural nucleotides | 20:27 |
| fenn | even infrared | 20:27 |
| kanzure | which significantly significantly enhances mutation | 20:27 |
| kanzure | mutation rates, I mean | 20:27 |
| kanzure | not mutation results | 20:27 |
| kanzure | oh, there's also the jellyfish stuff | 20:27 |
| kanzure | GFB, luciferase/luciferin, ? | 20:27 |
| kanzure | would really like a solid state laser ... but luciferin would be totally solid state | 20:27 |
| kanzure | blah, lots of labs have a list of fluoroproteins right? | 20:28 |
| kanzure | not focused/directed -- how much energy loss, noise, etc.? | 20:28 |
| kanzure | holding up a firefly is not like holding up a lamp | 20:28 |
| kanzure | maybe we can try making a huge mass of firefly luciferin | 20:28 |
| kanzure | as big as we can make it | 20:28 |
| kanzure | and just dump a huge bucket of luciferase on it and see if it lights up like a firecracker :) | 20:29 |
| fenn | sure, or just a couple drops | 20:29 |
| kanzure | how are we going to get that much luciferin | 20:29 |
| fenn | standard recombinant dna stuff | 20:29 |
| kanzure | it would require its own experiment before we even confirm this method though | 20:29 |
| fenn | to see if combining luciferin/luciferase generates light (quickly)? | 20:30 |
| kanzure | if it's accumulative | 20:30 |
| fenn | accumulative? | 20:30 |
| kanzure | i.e., will 5 grams of luciferin light up strong enough to signal the transcriptional circuit? | 20:30 |
| fenn | ah | 20:30 |
| fenn | probably not | 20:30 |
| fenn | :( | 20:30 |
| kanzure | bio is the only alternative to solid state that I'm going to accept here, I think. not sure. | 20:31 |
| kanzure | solid state advantages: it's freaking reliable (photons, lasers) | 20:31 |
| kanzure | bio advantage: just replicate more | 20:31 |
| fenn | solid state is also quite cheap these days | 20:31 |
| kanzure | replicate enough to do the synthesis you're planning for a few weeks in advance, or something | 20:31 |
| kanzure | also, DIY laser projects on the net | 20:31 |
| kanzure | and organic LEDs as another option.. | 20:31 |
| kanzure | but on the other hand, "go build a fire with sticks and stones" might work, right? | 20:32 |
| kanzure | http://en.wikipedia.org/wiki/Organic_light-emitting_diode | 20:32 |
| kanzure | oLEDs might be too manufacturing-specific (i.e., fabs-only) | 20:32 |
| fenn | so are LED's | 20:33 |
| kanzure | wouldn't it be nice if we could just have a pigment filter for a light source | 20:33 |
| kanzure | then we just need to figure out how to get enough candles / luminosity | 20:33 |
| kanzure | or some laser variable. | 20:33 |
| kanzure | intensity | 20:33 |
| fenn | what about sunlight/colored gel/shutter | 20:33 |
| kanzure | colored gel? | 20:33 |
| kanzure | hrm | 20:34 |
| fenn | to filter out the light | 20:34 |
| kanzure | oh, food coloring | 20:34 |
| kanzure | well shit. | 20:34 |
| fenn | its called a gel for some reason | 20:34 |
| kanzure | food coloring works very, very well | 20:34 |
| kanzure | it blocks out specifically that color | 20:34 |
| kanzure | so it's the absence of it | 20:34 |
| kanzure | that's ... close. | 20:34 |
| kanzure | would it be enough? | 20:34 |
| fenn | the thing is, your eye sees only one color at a time | 20:34 |
| fenn | but we really want monochromatic light | 20:34 |
| kanzure | hm? | 20:34 |
| fenn | and a messy spectrum with everything but green missing will look red | 20:35 |
| fenn | even though its not monochromatic red | 20:35 |
| kanzure | how is that possible? | 20:35 |
| fenn | er.. with green missing i mean | 20:35 |
| kanzure | everything-but-green ==> red? | 20:35 |
| kanzure | oh | 20:35 |
| kanzure | green-missing => red? | 20:35 |
| fenn | yeah | 20:35 |
| kanzure | is this something that I should be aware of already | 20:35 |
| kanzure | like 7th grade science or something? sounds very peculiar | 20:36 |
| fenn | hmm.. you know rods/cones, yah 7th grade science | 20:36 |
| fenn | look up color blindness | 20:36 |
| kanzure | okay | 20:36 |
| kanzure | are we talking about the human eye, or color input for any system | 20:36 |
| fenn | i'm just saying food coloring won't necessarily produce a tight spectrum | 20:37 |
| kanzure | oh, pigmentation purity issues | 20:37 |
| kanzure | is that it? | 20:37 |
| fenn | how about a prism or diffraction grating | 20:37 |
| kanzure | http://en.wikipedia.org/wiki/Diffraction_grating 'In optics, a diffraction grating is an optical component with a regular pattern, which splits light into several beams travelling in different directions. ' (huh, like a prism) | 20:37 |
| kanzure | hrm. | 20:37 |
| kanzure | crystals? | 20:38 |
| kanzure | how does one go about making a prism | 20:38 |
| kanzure | http://en.wikipedia.org/wiki/Prism_(optics) | 20:38 |
| fenn | a triangular chunk of something clear | 20:38 |
| kanzure | even gel? | 20:38 |
| fenn | sure | 20:39 |
| kanzure | can we grow gels? | 20:39 |
| fenn | i dont know how you'd get it into a triangular shape | 20:39 |
| kanzure | if it's stable, then physical sawing / sharp pointy stick | 20:40 |
| kanzure | how exact? | 20:40 |
| fenn | who's gonna saw it? | 20:40 |
| kanzure | me? | 20:40 |
| fenn | oh | 20:40 |
| fenn | in that case, why make it from gel | 20:40 |
| kanzure | oh, what if the gel grows in a triangular container? | 20:40 |
| fenn | you still need a triangular container | 20:41 |
| kanzure | actually, I've heard some terms recently | 20:41 |
| kanzure | apparently you prepare a gel in the lab | 20:41 |
| kanzure | by mixing some chemicals together | 20:41 |
| kanzure | are these chemicals, biological in origin? | 20:41 |
| kanzure | an ECM of some sort? | 20:41 |
| fenn | ECM? | 20:41 |
| kanzure | extracellular matrix | 20:41 |
| kanzure | re: needing the container; you need the tank too. | 20:41 |
| fenn | ok, where do you get the tank? | 20:42 |
| kanzure | scrap metal :( that's an unsolved problem | 20:42 |
| fenn | anyway, there's a big ugly complex electromechanical shutter mechanism to worry about too | 20:42 |
| fenn | granted, it could be just a motor with some fan blades | 20:42 |
| kanzure | easy system: cardboard paper + servo motor to spin it around + keep it in the path from light -> circuit; then, spin it around to open the gate | 20:43 |
| fenn | but those dont exactly grow on trees either | 20:43 |
| kanzure | yes | 20:43 |
| kanzure | right | 20:43 |
| kanzure | well, we mentioned an electrical shock | 20:43 |
| kanzure | which was to be used to do the bioluminescence | 20:43 |
| kanzure | in this case, however, we just need a shock to clear a 'buffer' or 'blocker' | 20:44 |
| kanzure | hrm, that sucks | 20:44 |
| fenn | you're thinking like a liquid crystal? | 20:44 |
| fenn | or a kerr cell (ignore) | 20:44 |
| kanzure | liquid crystal might work, how would we implement? | 20:44 |
| fenn | i think this project is somewhat lacking in rigorous specifications | 20:45 |
| kanzure | of course | 20:45 |
| fenn | i think its best to ignore the 99% of the rest of the technological infrastructure leading up to getting blinky lights | 20:47 |
| fenn | and just pretend you've got blinky lights | 20:47 |
| kanzure | oh | 20:48 |
| kanzure | I guess somebody else can come along and do it anyway | 20:48 |
| kanzure | the blinky lights aren't too important to the overall circuit really | 20:48 |
| fenn | the thing is, we dont have a direct write method for DNA yet | 20:48 |
| kanzure | remember, the frequency-input mechanism on the bio side isn't necessarily light-based | 20:48 |
| kanzure | it might be electrical if we want, or something, I'll have to ask the guys at the lab about this | 20:49 |
| kanzure | okay, so the DNA write method | 20:49 |
| fenn | ok | 20:49 |
| kanzure | http://heybryan.org/mediawiki/index.php/DNA_synthesizer | 20:49 |
| kanzure | I have some notes on the oligo synthesis reactions, but not much | 20:49 |
| kanzure | starting @ "The first step in the synthesis is the removal of the dimethoxytrityl (DMT) group with TCA to free the 5'-hydroxyl for the coupling reaction. The next step, activation, is achieved by simultaneously adding a phosphoramidite derivative of the next nucleotide and tetrazole, a weak acid to the reaction chamber." | 20:49 |
| kanzure | "The tetrazole protonates the nitrogen of the phosphoramidite, making it susceptible to nucliophilic attack. This intermediate is very reactive and the following coupling step is complete in less than 30 seconds. The 5'-OH group of the phosphoramidite is blocked with the DMT group." | 20:50 |
| fenn | blah | 20:50 |
| fenn | i dont care | 20:50 |
| fenn | that's chemical engineering bullshit | 20:50 |
| kanzure | but how would we build a DNA molecule | 20:51 |
| kanzure | if we could make it think that it's building the complementary strand, that'd be great | 20:51 |
| kanzure | it == transcription factors and so on | 20:51 |
| kanzure | erm, proteins related to the proc | 20:51 |
| fenn | you'd have one molecule to hold the base in place, and then ligase comes along and bonds it to the backbone | 20:51 |
| fenn | know how a line printer works? | 20:52 |
| kanzure | ink based? | 20:52 |
| kanzure | I know how a laser jet works | 20:52 |
| kanzure | the 'laser' fires and writes to the roller, the roller presses down on the paper | 20:53 |
| fenn | there's a wheel which spins around to the correct letter (nucleotide) and then it goes *wham* and hits against a piece of paper (ligation) | 20:53 |
| fenn | you can have a whole bunch of wheels spinning in parallel, so you print a whole line at a time | 20:53 |
| fenn | anyway, i'm thinking just one wheel | 20:54 |
| fenn | with 4 letters | 20:54 |
| kanzure | ok, when you say wheel that spins around to the correct nucleotide, I'm thinking casino "777 YOU WIN!!" spinners | 20:54 |
| kanzure | and it sounds like a nasty biomolecule ... | 20:54 |
| fenn | yeah something like that | 20:54 |
| fenn | actually it doesnt even need to ligate, just hold them in place while another molecule uses them as a template | 20:55 |
| fenn | dunno if that would really work though | 20:55 |
| kanzure | I don't like this. sounds like spooky molecular engineering. | 20:55 |
| kanzure | and the molecule *must* be replicable (i.e., be a protein) | 20:55 |
| fenn | ya | 20:55 |
| kanzure | hey, uh, to speed up transcriptonial switches, why not run them while in a centrifuge? | 20:56 |
| fenn | nope | 20:56 |
| fenn | speed is governed by concentration and temperature | 20:56 |
| kanzure | it's just diffusion that limits it, right? | 20:56 |
| kanzure | concentration doesn't matter if they're covering the same space more quickly | 20:56 |
| fenn | centrifuge just increases the pressure, which causes some equilibrium shifts (favoring larger molecules) | 20:57 |
| fenn | also it wreaks havoc with molecular machinery because everything gets clumped together at the bottom | 20:57 |
| kanzure | sucks | 20:57 |
| kanzure | I don't like your biomolecule idea really | 20:58 |
| kanzure | what we have is some molecular gates that can hold the 'state' of the system | 20:58 |
| kanzure | I mean, hold which nucleotide needs to be done next | 20:58 |
| fenn | so, in computer terms that's a register | 20:58 |
| kanzure | (there is obvious noise issues -- some might still be manufacturing the last state's nt, but just encode some selection experiment into it) | 20:58 |
| kanzure | yes | 20:58 |
| kanzure | so if you have a register, what do you connect to | 20:58 |
| kanzure | to specify the next nucleotide? | 20:59 |
| kanzure | usually the only way that the next nucleotide is specified is ... mutation | 20:59 |
| kanzure | I know it sounds weird, but think it through | 20:59 |
| kanzure | you have a dna molecule and the only way you change it is through mutation | 20:59 |
| kanzure | or recombination with another dna molecule | 20:59 |
| kanzure | (so we need a ghost dna molecule) | 20:59 |
| kanzure | anyway, so the 'next' nucleotide is basically set unless the machinery itself makes an error | 20:59 |
| kanzure | so we need to be able to engineer these errors to be subject to our will | 21:00 |
| kanzure | via the signaling system we have | 21:00 |
| kanzure | make sense? | 21:00 |
| fenn | hmm.. could use a genengineered DNA repair enzyme to 'repair' each nucleotide incorrectly (swap in what we want) | 21:00 |
| kanzure | how would it know what spot it is at | 21:00 |
| kanzure | also, dna repair enzymes do not move so slowly | 21:00 |
| fenn | perhaps it could be light activated to move forward one bp | 21:01 |
| fenn | and otherwise do nothing | 21:01 |
| fenn | so "prep - load guanine" then "swap" | 21:01 |
| kanzure | movement kinetics ? | 21:01 |
| kanzure | hrm | 21:01 |
| kanzure | needs to have a piece of the 'protein puzzle' missing before it can move | 21:01 |
| fenn | not so much different from what i was saying before, only now it's crawling along a dna strand | 21:02 |
| kanzure | and then we swap in the replacement component via light signaling (or whatever) | 21:02 |
| kanzure | but the problem is that the concentrations suck | 21:02 |
| fenn | oh, you can use a single stranded dna and just fill in the blanks | 21:02 |
| kanzure | hm? | 21:02 |
| kanzure | no no | 21:02 |
| kanzure | I mean the repair enzmye needs to have a "push here to go" button | 21:02 |
| kanzure | and we provide the push via a particle | 21:02 |
| kanzure | however, these particles are everywhere | 21:03 |
| fenn | i dont understand | 21:03 |
| kanzure | who's to say that another particle won't hit that same enzyme a few seconds later? | 21:03 |
| kanzure | and therefore make it do two bases when we only really meant one? | 21:03 |
| fenn | that's what i'm trying to avoid, hence the "swap" signal | 21:03 |
| kanzure | the problem is that the DNA repair enzyme only works at a certain rate, and we need it to be 'phase locked' to our switch signals | 21:03 |
| kanzure | hm | 21:03 |
| kanzure | how would the swap signal avoid that? | 21:03 |
| kanzure | oh, the signal is extra | 21:03 |
| kanzure | another extra signal on top of it all? | 21:03 |
| fenn | you'd give it enough time to get ready (bind to the correct nucleotide) before giving the swap signal | 21:04 |
| kanzure | (would be nice if the DNA repair enzyme had a transcriptional switch attached to it .....) | 21:04 |
| fenn | another signal (wavelength of light) | 21:04 |
| kanzure | okay, so the transcriptional circuit would be used to inhibit/activate the production of the first signal | 21:04 |
| kanzure | and then another light type for the swap signal after a certain amount of time | 21:04 |
| kanzure | (that amount of time is to let the switches do their thing and so on) | 21:04 |
| kanzure | the 'first signal' would specify what the molecules are looking for | 21:05 |
| fenn | i'm not so fond of transcriptional circuits really | 21:05 |
| fenn | i guess you could use the oscillator as a clock/swap signal | 21:05 |
| kanzure | I don't think you understand though .. it's all DNA | 21:05 |
| fenn | but it would have to be predictable to within some very small percent | 21:05 |
| fenn | or you'll get framing errors | 21:05 |
| kanzure | well, that's a flip-flop of what I was thinking of. I wanted the swap signal to be some light source; and then the other one to be due to switches | 21:05 |
| fenn | if the other one is due to switches, you have no data | 21:06 |
| fenn | just some silly on-off pattern | 21:06 |
| fenn | 01010101010101 | 21:06 |
| kanzure | remember the 2 bits? | 21:06 |
| kanzure | oh | 21:06 |
| kanzure | but if you have 2 bits then you could select different particles to send the enzymes | 21:06 |
| kanzure | the repair enzymes that are waiting for the 'signaler' as for what to fetch ext | 21:06 |
| kanzure | *next | 21:06 |
| kanzure | wait, is it a "what to fetch next signal" ? so the two signals are officially: (1) fetch-next, (2) swap | 21:07 |
| kanzure | *next fetch | 21:07 |
| kanzure | fetch and swap | 21:07 |
| fenn | if i weren't so lazy i'd make a blender animation of this thing | 21:07 |
| kanzure | if I didn't suck so much with blender, I'd make an animation of this thing | 21:07 |
| fenn | yeah, that too | 21:08 |
| kanzure | okay, so the fetch signal -- it would be really, really great if this was an switch template from the synthetic circuits | 21:08 |
| kanzure | Ellington has supposedly worked with mutated polymerases | 21:08 |
| kanzure | or invented them or something :) | 21:08 |
| kanzure | is it polymerase that we want? | 21:08 |
| fenn | well.. polymerase can have dozens of pieces, each with their own function | 21:09 |
| fenn | some polymerases are simpler than others | 21:09 |
| fenn | dna repair polymerase, for example pol-I, are usually simpler | 21:09 |
| kanzure | I don't like the 'binding complex on polymerase' thing ... seems kind of hard to engineer, since polymerase is, itself, a protein | 21:10 |
| kanzure | (for the fetch signaler) | 21:10 |
| kanzure | erm, so we need multiple pol-I molecules floating around | 21:10 |
| fenn | check | 21:10 |
| kanzure | different ones for different molecules, we don't want it to have to have internal logic | 21:10 |
| kanzure | not a big issue, just a note | 21:10 |
| fenn | hmm | 21:10 |
| kanzure | without the fetch signaler it shouldn't work | 21:11 |
| kanzure | but when it has the fetch signal, only those that fit the fetch signal will work | 21:11 |
| fenn | pol-I crawls along the dna strand one base at a time | 21:11 |
| kanzure | crap | 21:11 |
| fenn | that's useful because it means you dont lose your place | 21:11 |
| fenn | also, its guaranteed that the enzyme will be there | 21:11 |
| kanzure | ok, scratch that last stuff about multiple types of pol-I then | 21:11 |
| fenn | so you only have to wait until a free nucleotide comes along | 21:11 |
| kanzure | we can't flush it quickly enough | 21:12 |
| kanzure | what are the specs here? | 21:12 |
| fenn | reliability | 21:12 |
| kanzure | needs to be able to accept a fetch and swap signal | 21:12 |
| kanzure | is that about it? | 21:12 |
| fenn | yep | 21:12 |
| kanzure | the fetch signal will be any of 4 types | 21:12 |
| kanzure | the swap signal will tell it to move | 21:12 |
| kanzure | hm, doesn't it move thanks to ATP or something? | 21:13 |
| kanzure | I don't recall. | 21:13 |
| fenn | the nucleotide has "ATP" built into it | 21:13 |
| kanzure | http://en.wikipedia.org/wiki/DNA_polymerase_I | 21:13 |
| kanzure | oh | 21:13 |
| fenn | ATP is just adenosine with phosphate sticking off it | 21:13 |
| fenn | there's also GTP TTP CTP | 21:13 |
| kanzure | http://ellingtonlab.org/ ' For example, we have evolved RNA polymerases that can utilize modified nucleotides. Finally, we have extended our evolutionary approaches to whole organisms, and are attempting to evolve 'unnatural' E. coli (unColi) that can augment their genetic codes with unnatural amino acids.' | 21:14 |
| kanzure | so evolving RNA polymerase seems to be possible | 21:14 |
| kanzure | grr | 21:14 |
| kanzure | how do we evolve polymerase for such specific functions? | 21:14 |
| kanzure | (fetch, swap) | 21:15 |
| fenn | i dont see the point of that really | 21:15 |
| kanzure | increases rate of mutation | 21:15 |
| kanzure | thanks to 'new' nucleotide types | 21:15 |
| fenn | how do they specify the location of the new nucleotide? | 21:15 |
| kanzure | thus you can put some intense selective pressure on them | 21:15 |
| kanzure | hrm | 21:15 |
| kanzure | maybe he's not? | 21:15 |
| fenn | there's lots of ways to increase mutation | 21:15 |
| kanzure | radiation hurray | 21:15 |
| fenn | location-specific mutation is useful. also highly random mutation. most methods are somewhere in between | 21:16 |
| kanzure | ''DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, | 21:16 |
| fenn | anyway, it doesnt get us any closer to in-vivo DNA synthesis | 21:16 |
| kanzure | ' while the rate of synthesis by pol I averages only 20 nucleotides/second. ' | 21:17 |
| kanzure | v | 21:17 |
| kanzure | http://en.wikipedia.org/wiki/DNA_polymerase_I | 21:17 |
| fenn | DNA replication is highly optimized for speed, accuracy, and efficiency. not necessarily simplicity or hackability | 21:17 |
| fenn | 20bp/s is good enough | 21:17 |
| fenn | e. coli genome is like 4kbp right? | 21:17 |
| kanzure | wtf? sounds unlikely | 21:18 |
| kanzure | but useful | 21:18 |
| fenn | one would expect this to be an easy google search | 21:18 |
| kanzure | 5E6 | 21:19 |
| kanzure | http://www.genome.wisc.edu/aboutus.htm | 21:19 |
| fenn | hmm ok Mbp | 21:19 |
| kanzure | 4000 genes | 21:19 |
| kanzure | so you were off by a term | 21:19 |
| fenn | 12 hours | 21:19 |
| fenn | oop, 70 hours | 21:20 |
| kanzure | ok, at 20 nt/sec, that has to be incredibly in sync | 21:20 |
| fenn | wah | 21:20 |
| kanzure | unless we can slow it down | 21:20 |
| kanzure | hey, let's slow it down with a signal too | 21:20 |
| kanzure | let's throw in another light source | 21:20 |
| fenn | why is it slow in the first place? | 21:20 |
| fenn | ok, premature optimization, who cares how fast it is | 21:20 |
| kanzure | Paula de Lucia; John Cairns (Dec 1969). "Isolation of an E. coli Strain with a Mutation affecting DNA Polymerase". Nature 224 (5225): 1164-66. doi:10.1038/2241164a0. PMID 4902142. | 21:21 |
| kanzure | well, we do | 21:21 |
| fenn | december 1969, when everything happened | 21:21 |
| kanzure | because it has to be slow enough to let the switches do their magic :( | 21:21 |
| kanzure | although | 21:21 |
| kanzure | hrm | 21:21 |
| kanzure | the switches obviously complicate things, but I'm sure there's a way | 21:22 |
| kanzure | generally halting it is a good thing | 21:22 |
| kanzure | has to be light, there's no way a diffuse reactant can get to all of them in time | 21:22 |
| fenn | could induce strand separation by coding a bunch of C-G's | 21:23 |
| kanzure | huh? | 21:23 |
| fenn | to stop the enzyme, kick it away from the template strand | 21:23 |
| kanzure | so pad everything? | 21:23 |
| fenn | C-G binding is weaker than AT binding | 21:23 |
| kanzure | how would the sequence be 'recovered'? | 21:23 |
| kanzure | oh | 21:23 |
| fenn | see what i'm saying? | 21:23 |
| kanzure | sort of | 21:23 |
| kanzure | no, I don't see how it would work to our advantage | 21:24 |
| fenn | ok nevermind | 21:24 |
| kanzure | ATGCCTAGGTCGCGCGCGC<kill> | 21:24 |
| kanzure | but now you have C-G's | 21:24 |
| kanzure | instead of your sequence :( | 21:24 |
| fenn | right | 21:24 |
| kanzure | and if you endonuke it, too many fragments right? | 21:25 |
| fenn | you can predict where restriction enzyme will cut | 21:25 |
| kanzure | oh, so the buffer will be the sequence for endonuclease | 21:25 |
| kanzure | then you do an endonuclease cut | 21:25 |
| fenn | yeah they are very specific to certain sequences (5 bp or so) | 21:26 |
| kanzure | <your sequence><endonuclease seq><kick the polymerase off> | 21:26 |
| kanzure | then endonuke it | 21:26 |
| kanzure | but you lose the position | 21:26 |
| kanzure | wasn't that the point of polymerase? | 21:26 |
| fenn | isnt that the point? | 21:26 |
| fenn | you're done | 21:26 |
| kanzure | no | 21:26 |
| kanzure | that's for one base | 21:27 |
| kanzure | :( | 21:27 |
| fenn | oh, why kick the polymerase off? | 21:27 |
| kanzure | to stop the enzyme | 21:27 |
| fenn | wait, are you saying use transcriptional circuits to synthesize a new polymerase enzyme for each base? | 21:27 |
| kanzure | so that you can give the switches time to induce the building of the 'fetch' signaler molecules | 21:27 |
| kanzure | nono | 21:27 |
| kanzure | the circuits control the production of the 'fetch-next' molecules | 21:28 |
| fenn | ok so the 'fetch' molecules are something like tRNA | 21:28 |
| kanzure | shit | 21:28 |
| kanzure | that sounds right :) | 21:28 |
| kanzure | \o/ translation? | 21:28 |
| fenn | well, tRNA normally grabs amino acids | 21:28 |
| fenn | yes | 21:28 |
| kanzure | tRNA could be an easy byproduct of the switches, I think | 21:29 |
| fenn | or it could be a short aptamer | 21:29 |
| kanzure | how would you dehook whatever the aptamer catches, (dehooking -> give to polymerase) | 21:29 |
| fenn | i dont see the need for a separate fetch molecule? | 21:30 |
| kanzure | funny, Wikipedia page "Translation" is on linguistic translation. Even though bio translation occurs in trillions of cells *per organism*. | 21:30 |
| kanzure | well, the fetch molecule tells the polymerase what to fetch next | 21:30 |
| fenn | so its more like information transfer than molecule transfer | 21:30 |
| kanzure | sure | 21:30 |
| kanzure | the actual molecule doesn't matter | 21:30 |
| * fenn pictures floppy disks being shuffled around | 21:31 | |
| kanzure | (but the swap signal, that probably should be a molecule or something) | 21:31 |
| kanzure | huh? | 21:31 |
| kanzure | that's nearly before my time | 21:31 |
| fenn | hah | 21:31 |
| kanzure | but I do remember loading floppies into machines | 21:31 |
| kanzure | to install software. | 21:31 |
| kanzure | "insert next disk" | 21:31 |
| fenn | 'never underestimate the bandwidth of a station wagon loaded with tapes hurtling across the countryside' | 21:32 |
| kanzure | unless you refer to the concept of a stack/list/queue | 21:32 |
| kanzure | so we need to get the information to the polymerase molecule | 21:32 |
| kanzure | and we need it to act on this information somehow | 21:33 |
| kanzure | kind of like switching out a prosthetic hand | 21:33 |
| kanzure | a new hand for a different job (eating, versus playing catch with a kid) | 21:33 |
| kanzure | (I've heard that people used to do that with old prosthetic hands) | 21:33 |
| fenn | prosthetic hands is a pretty sad state of affairs | 21:34 |
| fenn | so, instead of switching hands and then looking around for them each time, why not have a "wheel" with all the hands you need on it already | 21:34 |
| kanzure | how do you tell the wheel to rotate, and how do you tell it to rotate a certain amount, and how do you know that you're not rotating the wheel multiple times? | 21:35 |
| fenn | the wheel rotates on its own, but gets locked into a certain position when some "solenoid" is on | 21:35 |
| fenn | except in this case the solenoid is a light-activated protein | 21:35 |
| fenn | so when there's no input, the wheel is just spinning due to brownian motion | 21:36 |
| kanzure | how do you make a molecular wheel | 21:37 |
| fenn | or, it could be any old binding protein that gets synthesized by your transcriptional switch | 21:37 |
| fenn | i suppose it doesnt need to be a wheel, just some free floating proteins in close proximity, held together with 'chains' | 21:38 |
| fenn | so you just write it all on one gene like we talked about earlier with the purification tag | 21:38 |
| fenn | the fetch molecule would then come along and pull everything together | 21:39 |
| kanzure | wha? | 21:39 |
| kanzure | chains? | 21:39 |
| fenn | ok, so, instead of throwing your prosthetic hand behind the couch after you're done eating, you keep all your hands tied to your belt | 21:39 |
| kanzure | do we have a belt? | 21:40 |
| fenn | yeah, its the polymerase | 21:40 |
| kanzure | it doesn't seem to work like that | 21:40 |
| fenn | "you" are the polymerase | 21:40 |
| kanzure | I mean, it has no reconfigurable 'hand joint' | 21:40 |
| fenn | wah | 21:40 |
| kanzure | i.e., in the prosthetic hand case, "you are the polymerase" -> "you're the guy with the hook for the hand" | 21:40 |
| kanzure | how would you even think about evolving that ? | 21:40 |
| kanzure | the reconfigurable joint | 21:40 |
| fenn | i'm gonna go look for some youtube animations of polymerase | 21:41 |
| kanzure | how does it do it anyway? | 21:41 |
| kanzure | yeah | 21:41 |
| fenn | i'm hoping for a nice animation like for atp synthase, but its unlikely | 21:42 |
| kanzure | needs a 'hand eject' function | 21:43 |
| fenn | this is cool, a little too fast though :) http://youtube.com/watch?v=49fmm2WoWBs&feature=related | 21:45 |
| fenn | also very nice http://www.youtube.com/watch?v=E8NHcQesYl8&NR=1 | 21:47 |
| kanzure | need to have a 'hand eject' function | 21:49 |
| kanzure | think of it as if you're causing neurotransmitter receptor sites in plasma membranes to stop working | 21:49 |
| kanzure | erm, to just float off | 21:49 |
| kanzure | like lifeboats | 21:49 |
| kanzure | problem with that is like asking for you to detach an ion channel :) an ion channel is a *channel* :) not a flagella-like molecular extension | 21:50 |
| fenn | another cool animation (ribosome) http://www.youtube.com/watch?v=Jml8CFBWcDs&feature=related | 21:56 |
| kanzure | http://scholar.google.com/ search 'polA polymerase reduced OR slow OR inhibited' | 21:58 |
| kanzure | 'DNA polymerase active site is highly mutable: Evolutionary consequences' | 21:58 |
| kanzure | http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=25787 | 21:59 |
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| kanzure | ' (motif A) of Thermus aquaticus DNA polymerase I. After selection, by using genetic complementation, we obtained a library of approximately 8,000 active mutant DNA polymerases, of which 350 were sequenced and analyzed. This is the largest collection of physiologically active polymerase mutants. We find that all' | 22:00 |
| kanzure | woah :) | 22:00 |
| fenn | meh | 22:00 |
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| fenn | patent avoidance search :) | 22:00 |
| kanzure | ' Bacteria dependent on these mutated polymerases for survival are fit to replicate repetitively. ' | 22:01 |
| kanzure | hellooo | 22:02 |
| kanzure | ' that bears the temperature-sensitive DNA polymerase I mutatation, polA12. The mutant enzyme has a reduced electrophoretic mobility and sedimentation rate. It is abnormally thermolabile and is rapidly inactivated at low salt concentrations.' | 22:02 |
| kanzure | salt is metallic, use quick magnets | 22:02 |
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| fenn | what we need is 80/20 for proteins | 22:06 |
| kanzure | ? | 22:06 |
| fenn | some basic folding motif that we can plug together like legos to build molecular structure | 22:06 |
| kanzure | folding chemistry consists of what? | 22:07 |
| fenn | instead of custom holistically-crafted kanji characters | 22:07 |
| fenn | something more like the roman alphabet | 22:07 |
| Vedestin | changing the physical structure of proteins | 22:07 |
| kanzure | Vedestin: would require protein folding, or lots of selection | 22:08 |
| kanzure | fenn is talking about something, else | 22:08 |
| Vedestin | oh is he? | 22:08 |
| kanzure | yes, 'instead of custom holistically-crafted kanji characters' | 22:08 |
| fenn | i'm trying to think of an analogy | 22:08 |
| kanzure | I thought the language analogy was mostly good | 22:08 |
| kanzure | but kanji is still object/pattern recognition | 22:09 |
| kanzure | there's nothing quite like the way that life is doing it | 22:09 |
| kanzure | well | 22:09 |
| kanzure | there has to be, it's physically occuring | 22:09 |
| fenn | the idea is to build a discrete set of modules that stick together, and design your protein machine based on those instead of trying to fold the whole thing like origami | 22:10 |
| kanzure | probably avoiding protein folding | 22:11 |
| kanzure | or if we do folding, it's only through mechanisms we know about | 22:11 |
| fenn | it throws the pol-I out with the bath water though | 22:12 |
| kanzure | of course | 22:12 |
| kanzure | it throws mostly everything out | 22:12 |
| fenn | i.e. i still dont know how to make a 'hand' | 22:12 |
| fenn | so, i think this is more than an evening's work | 22:12 |
| kanzure | well, we just need to hack polA | 22:13 |
| kanzure | pol I, whatever | 22:13 |
| kanzure | so I'm thinking a few ways: (1) selection experiments, mutations | 22:13 |
| kanzure | (2) biomolecular engineering with protein folding. let's hope we can model the folding of polymerase. | 22:14 |
| kanzure | and then we can do something computational like that | 22:14 |
| fenn | hack it how? | 22:14 |
| kanzure | we're obviously going to make a new polA, right? | 22:14 |
| kanzure | so we'll take the sequence and modify it | 22:14 |
| fenn | um, yes, but how do we know the correct sequence | 22:15 |
| kanzure | '… response of E. coli to low levels of alkylating agent: The role of polA in killing adaptation ' | 22:15 |
| kanzure | right, we have to figure out how to 'hack' it | 22:15 |
| kanzure | I bet somebody has mapped this | 22:15 |
| fenn | it's the old 'protein folding' problem | 22:15 |
| kanzure | let's say it has a seq of len 5 | 22:15 |
| kanzure | of len 50, actually | 22:15 |
| kanzure | so maybe bp 5 to 15 are what we are looking for | 22:15 |
| kanzure | if so, we're in super-duper luck | 22:15 |
| kanzure | the smaller the better :) | 22:16 |
| kanzure | we're looking for some way to control mutagenesis, remember | 22:16 |
| fenn | no, i could care less about mutagenesis | 22:16 |
| kanzure | so we need to be able to leave some sort of 'molecular hookon' jiggler that we can trigger when we have the polymerase molecule going about | 22:16 |
| kanzure | erm? the idea is to control when it 'makes an error' | 22:16 |
| kanzure | to hack the error process | 22:17 |
| kanzure | and just make that the default | 22:17 |
| fenn | it should always be making an error | 22:17 |
| kanzure | and be in control (grab life by the horns) | 22:17 |
| kanzure | heh, paper title: The Horns of Polymerase | 22:17 |
| fenn | ok, we have good control over the sequence, but no way to map that to a folded structure except through reverse engineering | 22:17 |
| kanzure | right, but lots of people have focused on polymerase already | 22:18 |
| kanzure | so we might have a 'map of polymerase mutations' | 22:18 |
| kanzure | and if anything on that map looks potentially useful | 22:18 |
| kanzure | then we're good | 22:18 |
| fenn | if we had a direct write tool, we could do lots of trial and error to figure out some motifs | 22:18 |
| kanzure | right | 22:18 |
| kanzure | so it might be one-to-one like that in the case of polymerase, it might not be | 22:18 |
| kanzure | it's something to investigate in the literature | 22:18 |
| fenn | but we dont have a direct write tool, so its kind of a dependency loop | 22:18 |
| kanzure | I have a lab | 22:18 |
| fenn | i think modern methods are too slow and labor intensive to get anywhere in a reasonable amount of time/money | 22:19 |
| fenn | $15/bp? well what if it takes a couple billion bp in trial/error to figure out some basic patterns | 22:19 |
| fenn | so, how many bp does it take anyway? :) | 22:20 |
| kanzure | $0.15/bp http://e-oligos.com/ | 22:20 |
| kanzure | hrm | 22:20 |
| kanzure | we would have to evolve it without sequence engineering :( | 22:20 |
| kanzure | and I'm clueless as to the design of selection experiments, where we want to add input/output | 22:21 |
| kanzure | to the target.. | 22:21 |
| fenn | veolve what? | 22:21 |
| * fenn kicks ssh in the nads | 22:21 | |
| kanzure | evolve our hacked polymerase | 22:21 |
| kanzure | oh, lag? | 22:21 |
| fenn | ping works without any delay, but for some reason my typing doesnt get echoed for like 20 seconds | 22:22 |
| kanzure | uh, also, how would we make polymerase work anyway? it needs a template strand to work with | 22:22 |
| kanzure | a template strand to copy, I mean. | 22:23 |
| fenn | u is small, can squeeze A G C T on the other side | 22:23 |
| fenn | er, uracil, sorry | 22:23 |
| kanzure | huh? | 22:23 |
| fenn | its the RNA version of thymine | 22:23 |
| kanzure | right | 22:24 |
| kanzure | but what does squeezing have to do with anything? | 22:24 |
| fenn | you wouldnt be copying the template strand, just using it to crawl along | 22:24 |
| fenn | the information comes from elsewhere | 22:24 |
| fenn | so its not actualy a template, more like a scaffold | 22:24 |
| fenn | so, when you add some arbitrary sequence opposite the scaffold strand, the bases dont really fit | 22:25 |
| fenn | normally the "doesnt really fit" would activate the proofreading domain | 22:26 |
| fenn | its kind of amazing we hav all this computer power and it isn't enough | 22:27 |
| kanzure | for typing? :) | 22:28 |
| fenn | for simulating protein, folding, and interactions between proteins | 22:29 |
| kanzure | re: protein folding, see CASP, the protein folding prediction compettition | 22:29 |
| fenn | say you have the sequence for pol-I, and the structure of DNA, you should be able to watch it go | 22:30 |
| fenn | like that program Phun | 22:30 |
| fenn | it doesnt have to correspond to reality 100%, just close enough that the same principles of design apply | 22:31 |
| * kanzure doubts we know protein engineering design principles :( | 22:31 | |
| fenn | well, we know the physical chemistry interactions between all the different amino acids | 22:32 |
| kanzure | interesting | 22:32 |
| kanzure | I just found a Nature review 'The descent of polymerization' | 22:32 |
| kanzure | I clicky, and it's Andy | 22:32 |
| kanzure | plus the last co-PI guy, Matt Levy | 22:33 |
| kanzure | 'The in vitro selection of a ribozyme polymerase capable of catalyzing the faithful addition of up to 14 nucleotides to a series of noncovalently bound primers fills a niche in our understanding of the origins and evolution of life on Earth.' | 22:33 |
| kanzure | A. Levskaya, A.A. Chevalier, J.J. Tabor, Z.B. Simpson, L.A. Lavery, M.Levy, E.A. Davidson, A.Scouras, A.D. Ellington, E.M. Marcotte, and C.A. Voigt (2005). Engineering Escherichia coli to see light. | 22:35 |
| kanzure | http://ellingtonlab.org/mediawiki-1.10.0/index.php/Main_Page | 22:36 |
| kanzure | guess I need to find a team that might do something related | 22:36 |
| kanzure | Team ITS; Eric -- Emulsion selections of polymerase control regions (Near-term priorities) | 22:36 |
| kanzure | Team Autogene; Eric -- Emulsion selections of polymerase enzymes Near-term priorities) | 22:36 |
| kanzure | sounds about right? | 22:36 |
| kanzure | haha, one guy. | 22:36 |
| kanzure | "Team ITS. Eric." | 22:36 |
| fenn | know any good protein visualization tools? | 22:38 |
| kanzure | yes | 22:39 |
| kanzure | no | 22:39 |
| kanzure | http://heybryan.org/mediawiki/index.php/Computational_biology | 22:39 |
| kanzure | http://heybryan.org/mediawiki/index.php/Comp_chem_linkdump | 22:39 |
| kanzure | the #bioinformatics people will, if that comp bio page doesn't | 22:41 |
| fenn | wonder if garlic can handle proteins well enough | 22:41 |
| kanzure | eh? | 22:41 |
| fenn | oh good, that's what it's for :) | 22:44 |
| fenn | i knew i was onto something | 22:44 |
| fenn | omg nice interface (not) | 22:45 |
| kanzure | hm? | 22:45 |
| kanzure | I'm going to email Eric | 22:45 |
| kanzure | how do I sound, not stupid | 22:45 |
| kanzure | guess I need to know what polymerase 'control regions' are | 22:45 |
| kanzure | are they really control regions | 22:45 |
| kanzure | ' | 22:48 |
| kanzure | Whether or not genes are in an active or a repressed state in a cell depends on the relative effect of gene silencers and locus control regions (LCRs). Here, we suggest that these elements act as binary switches; the state that prevails (activated or expressed) probably depends on a competition between protein complex formation and the stability of the complexes formed at either of the two elements.' | 22:48 |
| kanzure | oh, duh | 22:48 |
| kanzure | so apparently 'enhancers' in transcription are related to the binding sites | 22:49 |
| kanzure | http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2169052 Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis | 22:53 |
| kanzure | Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis 5 bp core subunit enzyme? | 22:55 |
| kanzure | erm | 22:55 |
| kanzure | http://www.pingrysmartteam.com/RPo/RPo.htm | 22:55 |
| kanzure | polymerase holoenzyme? | 22:55 |
| fenn | enhancers are regions of DNA before a gene that attract transcription factors | 22:55 |
| kanzure | ' In order to recognize a promoter to begin transcription, the 5-subunit core enzyme (α,α,β,β’,ω) must bind to one of various sigma (σ) factors; this form of the enzyme is called the holoenzyme. Each of the different σ factors recognize different promoter elements upstream of genes allowing the cell to respond to various environmental cues.' | 22:55 |
| kanzure | ''Once holoenzyme binds the promoter, DNA downstream of this interaction is brought into the enzyme and melted to expose single-stranded DNA. This stage of transcription initiation is described here using a model of RNA polymerase-open-promoter-complex (RPo)' | 22:55 |
| kanzure | so RNA polymerase-open-promoter-complex sounds important | 22:55 |
| kanzure | now if only we had this for DNA polymerase | 22:56 |
| kanzure | http://www.pnas.org/cgi/content/full/97/10/5095 DNA polymerase active site is highly mutable: Evolutionary consequences | 22:56 |
| fenn | why do you want that for DNA polymerase? (it exists, look up 'ori site') | 22:57 |
| fenn | (origin of replication) | 22:57 |
| kanzure | oh | 22:57 |
| kanzure | I already saw that paper | 22:57 |
| kanzure | well | 22:57 |
| kanzure | we want to find the complexes on the polymerase molecule | 22:57 |
| kanzure | and then see if anybody has reverse engineered this and shown that this correlates to an *exact* subset of the entire gene for polymerase | 22:57 |
| kanzure | "site-directed mutagenesis" | 22:58 |
| fenn | reverse engineered the mapping between genome locations and functionality in the polymerase? | 22:58 |
| fenn | or mapping between functionality and structural subunit in the polymerase? | 22:59 |
| fenn | or something else? | 22:59 |
| kanzure | the first | 22:59 |
| kanzure | http://www.pubmedcentral.nih.gov.ezproxy.lib.utexas.edu/articlerender.fcgi?artid=452606 | 22:59 |
| kanzure | ' Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. ' | 23:00 |
| fenn | ok that's easy. just knockout a particular nucleotide using "site directed mutagenesis" | 23:00 |
| kanzure | here's what I'm thinking of doing | 23:00 |
| fenn | an insertion will caluse a frame error | 23:00 |
| kanzure | we'll take a molecular dynamics simulation package of polymerase | 23:01 |
| kanzure | we'll then use this to just hack a way and add some extra functionality that we like | 23:01 |
| fenn | also you can do bacterial transformation stuff, its like reading a tape | 23:01 |
| kanzure | we'll then make some general simulations and predictions | 23:01 |
| kanzure | and then we'll design selection experiments that would find that | 23:01 |
| fenn | negatory | 23:02 |
| kanzure | hm? | 23:03 |
| fenn | molecular dynamics cant handle big roteins like polymerase, and on top of that, we can't hack in new functionality because we dont know how to do that | 23:03 |
| kanzure | 40,000 atoms on a 'top of the line' consumer-market machine | 23:03 |
| fenn | 40kD doing what? | 23:04 |
| fenn | looking pretty? or actually functinoning | 23:04 |
| kanzure | hrm, good question | 23:04 |
| kanzure | also, we have Big Iron available at the lab | 23:04 |
| fenn | with much red tape no doubt | 23:05 |
| kanzure | apparently not | 23:05 |
| kanzure | this one looks important - 'The Conserved Active Site Motif A of Escherichia coli DNA Polymerase I Is Highly Mutable' | 23:08 |
| kanzure | haha | 23:10 |
| kanzure | UmuC/DinB family (e.g. DNA polymerase ). Crystal struc- | 23:10 |
| kanzure | tures of representative enzymes from the first four families | 23:10 |
| kanzure | have been determined, revealing a common overall architec- | 23:10 |
| kanzure | ture that has been likened to a human right hand, with fingers, | 23:10 |
| kanzure | thumb, and palm subdomains (5–9). Although the structures of | 23:10 |
| kanzure | the fingers and thumb subdomains vary considerably, the cat- | 23:10 |
| kanzure | alytic palm subdomains are all superimposable (10, 11). The | 23:10 |
| kanzure | acid residue. Essential roles of motif A in catalysis include | 23:11 |
| kanzure | interaction with the incoming dNTP and coordination with two | 23:11 |
| kanzure | divalent metal ions that are required for the polymerization | 23:11 |
| kanzure | reaction (12–15). Motif A begins at an anti-parallel -strand | 23:11 |
| kanzure | hm. | 23:11 |
| fenn | here's a couple pics if it helps http://www.ncbi.nlm.nih.gov/Web/Newsltr/FallWinter02/structure.html | 23:15 |
| fenn | another one, not pol-I though http://www.biochem.umd.edu/biochem/kahn/teach_res/Overheads/T7DNAP.jpg | 23:15 |
| kanzure | btw, I'm not sure if looking at 3D visualizations 'helps' much | 23:19 |
| kanzure | I mean, I'm still surprised that enzymes sometimes have 'knooks and cubbies' for other things to connect into | 23:19 |
| kanzure | mainly because the mechanical probability of anything happening there seems small to me | 23:19 |
| kanzure | won't it be mostly chemical interactions at that scale? so shape won't matter much? | 23:19 |
| fenn | yes and no | 23:22 |
| fenn | its the moore's law bug | 23:23 |
| fenn | shape still matters even at the small molecule level | 23:23 |
| fenn | ever used a microscope at 1000X zoom? bacteria are bouncing around all over the place from brownian motion, so their insides must be just whirring around at a tremendous rate | 23:26 |
| kanzure | I like to imagine giant floating molecules of stuff from chem 101 interacting with other largely diffused molecules, and then radically changing shape | 23:27 |
| kanzure | but I guess shape can be conserved in some reactions | 23:27 |
| fenn | and the insides are not mostly water like they show in pictures, it's all really tightly packed in there.. i think its like 30% free water | 23:27 |
| fenn | ah the power of the internet:, i found it: http://mgl.scripps.edu/people/goodsell/illustration/public | 23:29 |
| kanzure | would it surprise you if I say I've seen that image before | 23:31 |
| fenn | no | 23:31 |
| fenn | i had it hanging on my wall for a couple years | 23:31 |
| kanzure | big? | 23:33 |
| fenn | scanned it from my mol-bio book and printed on a poster printer | 23:34 |
| kanzure | who the hell named this thing 'motif A' | 23:35 |
| kanzure | ooh | 23:36 |
| kanzure | catalysis (35). Both the N- and C-terminal parts of motif A | 23:37 |
| kanzure | tolerated a wide spectrum of substitutions. DNA polymerase | 23:37 |
| kanzure | activity associated with single amino acid substitutions within | 23:37 |
| kanzure | the N-terminal 5 amino acid residues was as high or higher | 23:37 |
| kanzure | than that of the wild-type enzyme. These residues form part of | 23:37 |
| kanzure | an anti-parallel -sheet structure that is believed to accommo- | 23:37 |
| kanzure | date the triphosphate moiety of the incoming dNTP and may be | 23:37 |
| kanzure | a potential target for engineering of pol I derivatives with | 23:37 |
| kanzure | altered properties. In contrast, amino acid substitutions within | 23:37 |
| kanzure | the C-terminal five residues tended to be associated with re- | 23:37 |
| kanzure | duced activity. | 23:37 |
| kanzure | "a potential target of engineering of pol I derivatives with altered properties" | 23:37 |
| kanzure | uh, yeah | 23:37 |
| fenn | http://www.inthenews.co.uk/news/science/scientists-launch-protein-folding-computer-game-$1222182.htm | 23:38 |
| kanzure | yep | 23:38 |
| kanzure | some 3D pathfinder or something | 23:38 |
| kanzure | "because people know how to do it better" etc etc | 23:39 |
| fenn | humans are supposed to be good at traveling salesman problems | 23:39 |
| kanzure | has this been tested? | 23:40 |
| kanzure | I see what you mean, but I'm wondering if we have some evidence | 23:40 |
| kanzure | both DNA and RNA substrates. We conclude that isoleucine at | 23:41 |
| kanzure | position 709 contributes to sugar discrimination by wild-type | 23:41 |
| kanzure | pol I and that this function may promote conservation of the | 23:41 |
| kanzure | wild-type motif A sequence. Based on analysis of a structural | 23:41 |
| kanzure | fun stuff, I think this paper is a goldmine | 23:41 |
| fenn | foldit looks really cool | 23:43 |
| kanzure | I need a second search box in Opera (yes, I'm back to Opera ... for the mean time) | 23:50 |
| kanzure | one that does Google Scholar directly | 23:51 |
| kanzure | with my proxy stuff already done | 23:51 |
| kanzure | Eric got back to me :( | 23:56 |
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