| --- Log opened Sun Jan 11 00:00:20 2015 | ||
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| kanzure | fenn: i think i have it, although it doesn't lookk exactly like what you want | 02:18 |
|---|---|---|
| kanzure | first, the most general approach is to use directed evolution by exposing cells to the conditions of oligonucleotide synthesis or something similar to it | 02:19 |
| kanzure | and those cells that increse yield will be the ones that you decide to keep for the next round | 02:19 |
| kanzure | which is quite similar to what you would be doing with enzymes anyway, except in a much more convenient package | 02:19 |
| kanzure | second, something about cell surface display of oligonucleotide-modifying enzymes, and then a collection of different cell types that you choose from for each step | 02:20 |
| kanzure | neither of these two strategies require a huge leap in rational design of protein or any other sort of magic | 02:21 |
| kanzure | although incorporating ration protein design guesses may speed along the process.... especially combinatorial mutation libraries related to cell surface display or something.. | 02:22 |
| kanzure | also, the cells do not need to come up with a solution related to surface display | 02:23 |
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| kanzure | nothing? | 05:14 |
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| kanzure | s/increse/increase | 05:50 |
| kanzure | s/ration protein design/rational protein design | 05:50 |
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| bbrittain | anyone know of a good 1080p dual LVDS laptop screen? preferably IPS, but optional. ~13 inches strongly prefered | 08:43 |
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| bbrittain | huh. ocassionally it's cheaper to order 5 things from china than to order one from a US supplier | 09:01 |
| chris_99 | bbrittain, have a look for the retina ones | 09:02 |
| yoleaux | 29 Dec 2014 02:46Z <nmz787> chris_99: latest CAD stuff https://github.com/nmz787/microfluidic-cad/blob/master/implicitCAD/output/tobacco_mesophyll_protoplast_fusion_device.svg | 09:02 |
| yoleaux | 29 Dec 2014 03:14Z <nmz787> chris_99: bit easier to see, but not as crisp: https://github.com/nmz787/microfluidic-cad/blob/master/implicitCAD/output/tobacco_mesophyll_protoplast_fusion_device.jpg | 09:02 |
| yoleaux | 29 Dec 2014 03:23Z <nmz787> chris_99: here is a snapshot of the 50MB STL, not as good as the SVG rendition, which wasn't perfect itself https://raw.githubusercontent.com/nmz787/microfluidic-cad/master/implicitCAD/output/tobacco_mesophyll_protoplast_fusion_device__3D.png | 09:02 |
| chris_99 | i think they're cheap | 09:02 |
| chris_99 | cheers nmz787! | 09:03 |
| bbrittain | chris_99: they unfortunatly use eDP connectors :( | 09:04 |
| chris_99 | oh darn | 09:04 |
| bbrittain | I don't really want to build a dual channel LVDS -> 4channel eDP board | 09:05 |
| bbrittain | that sounds like a pain in the ass | 09:05 |
| chris_99 | mmm | 09:05 |
| chris_99 | what are you making? | 09:05 |
| bbrittain | A laptop :) | 09:05 |
| chris_99 | ooh | 09:05 |
| chris_99 | i saw a thing on HN recently about making a projector which looks fun | 09:05 |
| bbrittain | I think I found a decent panel btw | 09:05 |
| chris_99 | linky? | 09:05 |
| bbrittain | http://www.amazon.com/B131HW02-LT131EE11000-VPC-Z-SERIES-Laptop/dp/B00HME9W1A/ref=sr_1_3?ie=UTF8&qid=1420995835&sr=8-3&keywords=LT131EE11000 | 09:06 |
| bbrittain | although I think I'll buy them for $20 each from china | 09:06 |
| bbrittain | :P | 09:06 |
| chris_99 | mm hehe | 09:06 |
| bbrittain | current progress on laptop is... little in way of concrete stuff. https://github.com/cavedweller/makemake | 09:07 |
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| chris_99 | how are you gonna make a case? | 09:08 |
| bbrittain | I have a 3d model I was building, but I'm a little worried about size and volume | 09:08 |
| bbrittain | I might use some solid acrylic or even carbon fiber, and then 3d print smaller components | 09:09 |
| chris_99 | cool | 09:09 |
| bbrittain | woohoo. bought the screen. all the components are gonna arive in the next few days :D \^o^/ | 09:13 |
| chris_99 | nice! | 09:15 |
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| fenn | bbrittain: i'm also building a laptop, but i started with a "lapdock" that has input ports, keyboard, battery | 09:39 |
| kanzure | fenn: thoughts about my surface display directed evolution plan? | 09:40 |
| fenn | i havent figured out power management though.. 19V is kinda annoying | 09:40 |
| fenn | kanzure: why surface? | 09:40 |
| kanzure | because you can do stepwise "chemistry" | 09:41 |
| kanzure | by physically removing the cells | 09:41 |
| kanzure | and inserting another cell with other surface display stuff | 09:41 |
| kanzure | the cells that help improve yield will over time be selected more than those cells that do not help yield | 09:41 |
| fenn | and you need 4^3 different cell types? | 09:42 |
| kanzure | one nucleotide at a time, so three cell lines at minimum | 09:43 |
| fenn | not four? | 09:43 |
| kanzure | oh yeah | 09:43 |
| kanzure | four... fine. | 09:43 |
| kanzure | well, you might also need capping/decapping, but hopefully you can coerce the cell lines into doing that for you | 09:44 |
| fenn | so these are just fancy bead-bound enzymes | 09:44 |
| kanzure | yeah, except they are self-replicating bead-bound enzymes | 09:44 |
| fenn | you could add magnetite genes for extra magic points | 09:44 |
| kanzure | they are self-replicating bead-bound enzymes that you don't have to sequence and re-synthesize after every time you find one doing something interesting | 09:45 |
| kanzure | instead you just don't flush it away into garbage | 09:45 |
| fenn | how do you make sure they only add one nucleotide at a time? | 09:45 |
| kanzure | you don't | 09:45 |
| kanzure | you could start with oligonucleotide synthesis, and just "add cells" | 09:46 |
| fenn | AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA | 09:46 |
| kanzure | those cells which make the reaction have higher yield are the cells that you decide to keep, and the others you flush | 09:46 |
| fenn | sure but eventually you want to make a specific sequence | 09:46 |
| kanzure | so, let's say you have some cells that help with the yield just a little bit | 09:47 |
| kanzure | over time you would select for those cells that help improve yield and also continue to do so in the presence of a diminishing supply of the chemical inputs | 09:47 |
| fenn | this sounds difficult to select for, but continue | 09:48 |
| kanzure | well it's easier to select than "find me a protein that responds correctly to all forms of light stimulation" since there's no known way to get from "light shining on a protein" to "correctly incorporating nucleotides and synthesizing a genome" | 09:48 |
| kanzure | anyway, each cell line will be evolved to (1) help decap and (2) help add a new nucleotide (specific to the cell line) | 09:49 |
| fenn | maybe easier to do deprotection with chemistry | 09:50 |
| kanzure | sure | 09:50 |
| fenn | or have 5 cell types | 09:50 |
| fenn | chemistry is faster since you can wash and deprotect at the same time | 09:50 |
| kanzure | no you can't? | 09:50 |
| fenn | ok maybe not | 09:51 |
| fenn | how about thermal deprotection | 09:51 |
| fenn | kills the enzyme at a lower temperature than the deprotection chemistry | 09:51 |
| fenn | then just keep adding stuff and zapping it | 09:52 |
| kanzure | shrug, whatever | 09:52 |
| fenn | anyway the chemistry will need to be designed before doing lots of selection/evolution work | 09:52 |
| kanzure | certainly | 09:53 |
| kanzure | in general i think that using cells is going to be easier than using enzymes directly | 09:53 |
| kanzure | because with enzymes you don't have an easy way to get the sequence that generated that enzyme | 09:54 |
| kanzure | whereas cells are self-contained genome packages | 09:54 |
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| fenn | yeah there's also protein-bound mRNA | 09:54 |
| kanzure | eh.. | 09:54 |
| * fenn shrugs | 09:54 | |
| kanzure | then you have to sequence the mrna | 09:54 |
| kanzure | and find what dna was responsible for that mrna | 09:55 |
| kanzure | and enzyme purification.... | 09:55 |
| kanzure | i'm not sure what cell/dna interactions are like, they might release nucleases that cut up dna | 09:56 |
| kanzure | in which case you would first have to run some selection projects to get cells that er, don't do that | 09:56 |
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| kanzure | i haven't really seen anything like "take a chemical process and just add cells and see which cells make the reaction better" before | 09:58 |
| kanzure | which i am a little suspicious about | 09:58 |
| kanzure | because people do that with enzymes.... so why not cells? | 09:58 |
| kanzure | i think people have been randomly testing enzymes in random chemical reactions to see how the enzymes work | 09:59 |
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| fenn | because most enzymes are not secreted by the cell | 10:07 |
| kanzure | that's true, but that's the cell's problem, not mine | 10:08 |
| kanzure | the cells which are more secretional will be selected for more than the other types | 10:08 |
| kanzure | or those cells with more protein structures on their surface that help with the reaction will be selected more than the other cells | 10:09 |
| kanzure | once you have those five cell types, you could then work on getting ones that can incorporate different nucleotides based on the presence of certain light signals. this could also involve some rational engineering of some proteins, if the mechanism that the cells are using is known/discovered.. and then over time you can select for a cell type that can respond to light correctly for discrimination between two possible nucleotides, and ... | 10:10 |
| kanzure | ... then a third, and finally a fourth light signal... | 10:10 |
| kanzure | light is ideal but in the mean time just being able to mechanically move the cells into position is probalby fine | 10:10 |
| kanzure | *probably | 10:11 |
| kanzure | the cells might find some local optima that cannot be easily rationally protein engineered to include conformational changes in response to light, e.g. might be multiple proteins or expression factors all at once, in which case you're a little bit screwed (maybe just keep running the experiment until a batch of cells is doing it using a single enzyme rather than multiple factors) | 10:12 |
| kanzure | but if you have these cells doing their jobs very efficiently then you might not need light anyway.... | 10:12 |
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| nmz787 | bbrittain: asus or acer has a dual screen laptop | 11:24 |
| nmz787 | bbrittain: http://www.asus.com/Notebooks_Ultrabooks/ASUS_TAICHI_21/ | 11:28 |
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| archels | but you can't really use them at the same time, in any useful sense :( | 11:31 |
| archels | I think a bunch of Macbook screens lend themselves well to hacking | 11:32 |
| archels | you might shoot your 1080p budget, though | 11:32 |
| nmz787 | bbrittain: I realize you wanted dual LVDS not dual screen | 11:33 |
| nmz787 | archels: you can | 11:33 |
| nmz787 | archels: you can do mirror or extend mode | 11:33 |
| archels | yeah but you can never seem them both at the same time, right? | 11:34 |
| archels | and it's always going to be angled wrong for either you or the person sitting opposite from you | 11:34 |
| nmz787 | the point is for biz presentations and such | 11:34 |
| nmz787 | IPS screens | 11:34 |
| nmz787 | so angle doesn't matter as much | 11:34 |
| chris_99 | cheers for those links nmz787 | 11:35 |
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| nmz787 | np, lately I've been thinking BRL-CAD might be the way to go for high-quality and open-source ability.... it is crazy that no one has BRL-CAD examples on github though... all their examples are buried in PDF files | 11:37 |
| chris_99 | odd | 11:37 |
| nmz787 | yup | 11:38 |
| chris_99 | ordered this yesterday - http://www.aliexpress.com/store/product/TEC-semiconductor-refrigeration-water-cooled-air-conditioning/709547_733123364.html | 11:39 |
| chris_99 | plus some h-bridge modules for it | 11:39 |
| nmz787 | hmm, doesn't seem like a terrible price | 11:41 |
| nmz787 | I got some pneumatic valves from aliexpress recently | 11:42 |
| chris_99 | cool | 11:42 |
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| nmz787 | they're kind neat, not what I thought... they're more like electrically-controlled pneumatic amplifiers... as they use the solenoid coil to open a small valve which let's your compressed air through, which then pushes a piston to switch the valve state (the piston is springloaded so when you turn off the coil, the air pressure behind the piston leaks through a small bleeder hole, and the spring pushes the piston back to the other position) | 11:43 |
| chris_99 | whatcha using them for? | 11:44 |
| nmz787 | microfluidic valving | 11:45 |
| chris_99 | ah cool | 11:45 |
| chris_99 | i need to order those microfluidic chips when i've got some cash | 11:45 |
| kanzure | nmz787: any opinions about the idea regarding directed evolution of extracellular cell-assisted oligonucleotide synthesis? | 12:26 |
| kanzure | see top of logs of http://gnusha.org/logs/2015-01-11.log | 12:27 |
| kanzure | yashgaroth: i guess you too | 12:28 |
| yashgaroth | I'm guessing this would be with TdT-based synthesis? | 12:40 |
| yashgaroth | "<fenn> AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA" mostly covers it | 12:43 |
| nmz787 | kanzure: I read your comments in today's log, I didn't make much sense of it really | 12:43 |
| nmz787 | heading out | 12:44 |
| kanzure | yashgaroth: actually i have no idea | 12:45 |
| kanzure | yashgaroth: frankly i don't strongly care how the cells do it, as long as they help somehow overtime | 12:46 |
| bbrittain | nmz787: I'm not a "cad person", but when I searched for tools during this project, what spoke to me was OpenSCAD | 12:46 |
| kanzure | yashgaroth: cells that are more helpful to oligonucleotide synthesis are more likely to be kept around by me, while i flush the less useful critters. | 12:46 |
| yashgaroth | directed evolution has a lot of foibles, I guess...cells are assholes so they're constantly trying to eat and/or degrade anything in their environment, but they're also annoyingly fragile, even moreso when you're applying a mutagen or w/e to speed up the evolution | 12:50 |
| kanzure | right... first task might be "get cells that do not degrade dna" | 12:50 |
| kanzure | er, and do not degrade oligos | 12:51 |
| kanzure | you can probably select for this too | 12:51 |
| kanzure | put some cells in with oligos, run gels | 12:51 |
| kanzure | shorter oligos than when you started means the cells are killingz the oligos | 12:52 |
| yashgaroth | erm theoretically yes | 12:53 |
| yashgaroth | it'd just be an enormous undertaking I guess | 12:54 |
| kanzure | elaborate on enormoose? | 12:55 |
| yashgaroth | well, either pressure-selecting cells that don't degrade your oligos, or modifying them to remove nucleases, keeping the whole thing sterile, what kind of cells you're using and the difficulty in growing them and displaying the enzymes you want, selection of said enzymes, mutation of those enzymes, in all likelihood introducing cells in has a far greater chance of making things go wrong | 12:57 |
| kanzure | presumably in the case of using cells, you wouldn't necessarily know which enzymes they come up with | 12:58 |
| kanzure | without studying the results later | 12:58 |
| kanzure | so you might just apply random mutagenesis if you had to 0~you might not have to though1~ | 12:58 |
| AmbulatoryCortex | how would you actually apply selection pressure to produce a certain enzyme, though? | 12:59 |
| kanzure | you aren't | 13:00 |
| kanzure | i don't care if they use enzymes or not | 13:00 |
| kanzure | it could just be some other byproduct for all i care | 13:00 |
| AmbulatoryCortex | well, same deal applies | 13:01 |
| AmbulatoryCortex | it's advantageous for them to eat dna, how would you change that? | 13:01 |
| AmbulatoryCortex | put in poison dna, maybe? | 13:01 |
| AmbulatoryCortex | but what would that be? | 13:02 |
| yashgaroth | chemo drugs, technically | 13:02 |
| yashgaroth | what about some directed evolution on TdT to get it to recognize the protected dNTPs, also work better at directed synthesis | 13:04 |
| yashgaroth | I know I have my concerns about the TdT stuff but if aqueous synthesis makes microfluidics a lot easier, it's a hell of a lot easier than anything with cells | 13:05 |
| kanzure | hmm, yes i think my cell proposal would work better with aqueous lifeforms | 13:20 |
| yashgaroth | I mean TdT being easier than cells in general, but yes aqueous tends to be more hospitable to both | 13:22 |
| kanzure | buuuut | 13:23 |
| kanzure | the tdt thing requires specific magic engineering right? | 13:23 |
| kanzure | whereas cell selection requires a billion cells and microfluidics automatically checking them maybe | 13:23 |
| yashgaroth | the magic would be to make it resistant to boiling like the cooper union people want; I just mean in general to make it more accurate/better processivity/etc | 13:26 |
| kanzure | oh then yes that is a million times easier | 13:26 |
| kanzure | well, wait | 13:26 |
| kanzure | no... you still have to trace the tdt back to which cell / genome / mRNA / DNA produced it... | 13:26 |
| yashgaroth | yeah but that's fairly standard art, dna display or whatevs | 13:28 |
| kanzure | speaking of which, i bet you could find a more stable tdt by searching some database and looking for extremophiles then diffing the residues to see wtf is different 0~if anything1~ in the different tdts | 13:28 |
| kanzure | but isn't dna display a lot of work | 13:28 |
| kanzure | especially the recovery process? | 13:28 |
| kanzure | and how do you know which tdt was working better | 13:29 |
| kanzure | just the ones that weren't degraded? :/ | 13:29 |
| yashgaroth | I doubt there's any TdT homolog present in lower lifeforms, since it's for immune diversity | 13:29 |
| kanzure | what's the process for reading the dna tags? just sequence and any sequences you get back you know were related? 0~also why wouold these tags not interfere with tdt function?1~ | 13:30 |
| kanzure | argh my keyboard is not giving me parens... i think it's my terminal or something, actually. | 13:30 |
| yashgaroth | well ideally you'd be recycling the protein in each well, otherwise you'd have to make a lot more, but it's not a huge problem | 13:30 |
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| yashgaroth | dna display is a lot less developed than phage display, but you can get one clone per well pretty easy | 13:30 |
| kanzure | ok so identification by wells.. then do a 1024x1024 plate.. hrm. | 13:30 |
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| kanzure | so you have to produce 1024x1024 different strains upfront? | 13:31 |
| kanzure | i guess i should look into the actual efforts required for mRNA/sDNA TAGGINGDFDF DDSSDFGDFAD ~~~ | 13:32 |
| yashgaroth | however many you want I guess, I mean if cambrian's single-bead synthesis actually works you can scale that to get one clone per well | 13:32 |
| yashgaroth | uhhhh | 13:32 |
| kanzure | sorry.. keyboard problems. | 13:33 |
| yashgaroth | I figured that or seizure | 13:33 |
| kanzure | a little bit of all three columns | 13:33 |
| kanzure | what are the limits of mRNA/DNA tags on proteins? | 13:34 |
| kanzure | like how long can these sequences be? can they be the full sequence 0~so that one-pot can work1~? | 13:34 |
| kanzure | gah still no parens? | 13:34 |
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| yashgaroth | I think so, haven't really looked into specific use cases | 13:35 |
| kanzure | i imagine it may be similar to antibody selection things | 13:35 |
| kanzure | antibody plates.. there are antibody plates, right? | 13:35 |
| kanzure | ah right you don't need to know their sequences, nevermind | 13:36 |
| yashgaroth | it's a lot more hassle than just finding strong antibody binders, which is what display tech is really designed for, but actually you don't really need to display per se | 13:39 |
| yashgaroth | just throw microfluidics in there somehow, done | 13:40 |
| kanzure | perfect | 13:41 |
| kanzure | i'll fax you your nobel prize later today, please have your phone line unoccopied | 13:41 |
| kanzure | *unoccupied | 13:42 |
| kanzure | er, which specific display technique is the mRNA/ssDNA one called? | 13:43 |
| kanzure | .wik mRNA display | 13:43 |
| yoleaux | "mRNA display is a display technique used for in vitro protein, and/or peptide evolution to create molecules that can bind to a desired target." — http://en.wikipedia.org/wiki/MRNA_display | 13:43 |
| yashgaroth | ^ | 13:43 |
| yashgaroth | with this it'd be more seeding ~1 mutated copy per well, let pcr do some work, then add cell-free ribosome stuff | 13:44 |
| kanzure | "The process results in translated peptides or proteins that are associated with their mRNA progenitor via a puromycin linkage" | 13:45 |
| kanzure | "Puromycin is an antibiotic that is a protein synthesis inhibitor by inhibiting translation." | 13:46 |
| kanzure | "Also of note, puromycin is critical in mRNA display. In this reaction, a puromycin molecule is chemically attached to the end of an mRNA template, which is then translated into protein. The puromycin can then form a covalent link to the growing peptide chain allowing the mRNA to be physically linked to its translational product." | 13:46 |
| kanzure | i thought amino acid sequences go out of a pore different from the mRNA exit pore | 13:47 |
| yashgaroth | I imagine it happens at the interface | 13:48 |
| kanzure | mRNA display and ribosome display overview http://www.bioc.uzh.ch/plueckthun/pdf/APpub0250.pdf | 13:58 |
| kanzure | bottom of page 13 has some speculation about how mRNA display works | 14:02 |
| kanzure | "In mRNA display, the purification of protein – puromycin –DNA –mRNA adduct from the ribosome presents a topological puzzle. After translation, the protein folds outside of the ribosomal tunnel to a globular domain. At the other end of the tunnel, the puromycin –mRNA/DNA reagent reacts with the polypeptide. Thus a folded domain sits at one end of the tunnel, while the long mRNA/DNA is connected to the peptide at the other end. ... | 14:03 |
| kanzure | ... Whereas the purification is performed under conditions expected to dissociate the ribosome, no direct evidence is yet available that an ‘‘opening’’ of the tunnel takes place. Alternative explanations are that (i) the mRNA/DNA passes through the protein exit tunnel, or (ii) the protein denatures and goes ‘‘backwards’’ through the tunnel. If such denaturation of the displayed protein is required, that might limit the ... | 14:03 |
| kanzure | ... application of mRNA display to proteins with robust refolding properties." | 14:03 |
| kanzure | "An interesting recent development are the so-called antibody mimics, proteins not closely related to antibodies that are designed to perform the antibody function of tight and specific target binding. A fibronectin type III domain, which has an immunoglobulin-like fold but is considerably smaller than a VH domain, was used to make mRNA-display libraries with diversity concentrated in three exposed loops; after selection Fn3-like domains ... | 14:06 |
| kanzure | ... with affinities as low as 20 pM were selected. An alternative approach employs proteins larger than antibody domains: ankyrins, proteins containing several repeats, have been used to create libraries with extensive randomized surfaces, and were subjected to ribosome-display selection to obtain high-affinity reagents. Without affinity maturation, low nanomolar KD values are normally obtained directly, leaving room for significant ... | 14:06 |
| kanzure | ... affinity improvement. Neither Fn3 domains nor ankyrins contain free cysteines or disulfide bonds; as a consequence, they perform better than most antibody fragments in bacterial expression and can be used under both oxidizing and reducing conditions. We anticipate that in-vitro-selected antibody mimics and antibody domains will become invaluable tools in proteomic research, as well as in biotechnology and in medical applications." | 14:06 |
| yashgaroth | magic, got it | 14:08 |
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| kanzure | those seem to be called "ankyrin repeat (AR) proteins" | 14:09 |
| kanzure | i'm glad someone tried that out | 14:09 |
| kanzure | you can just maximize surface area and surface binding variation per protein and then when you find something that works, just stress for deletions that don't delete the function you're interested in | 14:11 |
| kanzure | and then you magically get a smaller protein with that single (or a few) surface recognition sites | 14:11 |
| kanzure | well... fewer. | 14:11 |
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| kanzure | .wik snuffle cipher | 18:05 |
| yoleaux | "Bernstein v. United States is a set of court cases brought by Daniel J. Bernstein challenging restrictions on the export of cryptography from the United States." — http://en.wikipedia.org/wiki/Bernstein_v._United_States | 18:05 |
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| kanzure | .title http://www.unz.com/gnxp/the-2000-whole-genome/ | 18:39 |
| yoleaux | The $2,000 “whole genome” - The Unz Review | 18:39 |
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| kanzure | http://sakurity.com/blog/2015/01/10/hacking-bitcoin-exchanger.html | 20:16 |
| kanzure | "There’s a gaping hole in SmsAuthsController - two_factor_required! is only called for show action, but not for update which is actually responsible for activating SMS 2FA." | 20:21 |
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| delinquentme | That is a closure. A function doesn't have to return in order to be called a closure. Simply accessing variables outside of your immediate lexical scope creates a closure. | 22:40 |
| delinquentme | Is this correct? | 22:41 |
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