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| nmz787 | kanzure: as far as I can tell, the reason the thermo-labile ddNTPs isn't such a great idea is that they are expensive or unavailable, this seems to be their reference on terminators: http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=22570423 | 00:02 |
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| nmz787 | http://nar.oxfordjournals.org/content/40/15/7404.full.pdf | 00:03 |
| nmz787 | http://lasergen.com/technology/lightning-terminators/ | 00:04 |
| nmz787 | no prices | 00:04 |
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| nmz787 | I wonder if there is a thermo-regulatable tDt... chill the reactor then pulse with a laser/resistive-heater fast enough to incorporate only one unit | 00:16 |
| nmz787 | fenn: have you seen any reasonably priced screw + rail CNC kits? The laser etcher gantry pricelist is just under $500, most of which is a mcmaster acme screw and compensating wear nut | 01:56 |
| nmz787 | fenn: oO $15 for a 16" round MIC-6 plate http://www.sandsmachine.com/alumweb.htm | 02:12 |
| nmz787 | idk if this would be useful in some way http://www.aliexpress.com/store/product/New-Super-directional-speaker-kit-sharp-directivity-by-using-ultrasound-sound-only-straight/538835_32243617143.html | 02:39 |
| nmz787 | .title | 02:39 |
| yoleaux | Aliexpress.com : Buy Free shipping Jet 1300 C Butane Lighter from Reliable Lighters suppliers on Open source hardware | Alibaba Group | 02:39 |
| nmz787 | psh | 02:39 |
| nmz787 | just read the link | 02:39 |
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| nmz787 | hmm, http://deepblue.lib.umich.edu/bitstream/handle/2027.42/86196/ME450%20Fall2009%20Final%20Report%20-%20Project%2002%20-%20Maskless%20Photolithography%20System.pdf?sequence=1 | 02:55 |
| nmz787 | http://diyhpl.us/~nmz787/pdf/Mask-less_Photolithography_System.pdf | 02:55 |
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| nmz787 | fenn: ^ they used the same acme screw as you chose... except for a rotary table design, not XY | 03:02 |
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| nmz787 | they said they couldn't figure out how to hack a bluray drive, basically | 03:02 |
| nmz787 | the sponsoring prof does neural bio-mems http://www-personal.umich.edu/~chronis/ | 03:06 |
| nmz787 | paperbot: http://www.nature.com/nmeth/journal/v5/n6/pdf/nmeth.1203.pdf | 03:09 |
| paperbot | http://libgen.org/scimag/get.php?doi=10.1038%2Fnmeth.1203 | 03:09 |
| nmz787 | supplementary material http://www.nature.com/nmeth/journal/v5/n6/extref/nmeth.1203-S1.pdf | 03:09 |
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| nmz787 | the origin of the chip that kanzure like's to cite http://web.stanford.edu/group/foundry/services/PredesignedChips/chemostat_science05.pdf | 03:24 |
| nmz787 | and the supplement http://www.che.caltech.edu/groups/fha/publications/balagadde_supplemental.pdf | 03:26 |
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| nmz787 | kinda neat http://www.che.caltech.edu/groups/fha/publications/predator.pdf | 03:29 |
| nmz787 | fenn: I was able to implement a sine wave LUT in an arduino to set the PWM freq of a 28byj-48 stepper motor with uln2003 driver... to get high-bit precision microstepping, the difference I then found was that the fancier stepper controllers also have different decay modes which can make a decent difference if looking for smoothness, since the inertia and also I think reluctance or some sort of inductance smoothing electrical stuff out, at ... | 03:41 |
| nmz787 | ... least when a motor is turning in the same direction (reversing directions may benefit from higher bit microstepping, but idk really) | 03:41 |
| nmz787 | fenn: the 28byj-48 is geared down and already a high-step per revolution motor, so that may not even be an issue if it could be used... but I think the backlash and also speed make it suitable for only radial table designs, or really slow XY etching | 03:43 |
| nmz787 | I would think the same chopper concept should work with larger motors too, though larger drivers would likely be needed than the uln2003 | 03:43 |
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| nmz787 | microstepping with the polulu/allegro/easystepper chips should be fine though, since a single step with that acme screw and a 400step/rev motor is 1.5875 microns | 03:52 |
| nmz787 | lately my plan has been to make a macro-micro adapter with something like this, to interface with higher-res features made some other way (if/when needed) | 03:53 |
| nmz787 | that threaded rod is +- 15.24 micron per turn, seems pretty nice | 03:56 |
| nmz787 | dang vxb.com items went up by more than 20% | 03:58 |
| poppingtonic | the benbox engraver is pretty cool | 03:59 |
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| kanzure | nmz787: i'm becoming somewhat opposed to schemes involving light because of the extra steps it adds to the problem ("step zero: engineer a version of these enzymes to conformationally switch when exposed to photons"), especially considering that none of these polymerases are photoreactive by default | 06:02 |
| kanzure | "The Illustris project is a large cosmological simulation of galaxy formation ... We follow the coupled dynamics of DM and gas with the robust, accurate, and efficient quasi-Lagrangian code AREPO. In this approach, an unstructured Voronoi tessellation of the simulation volume allows for dynamic and adaptive spatial discretization, where a set of mesh generating points are moved along with the gas flow. This mesh is used to solve the ... | 06:41 |
| kanzure | ... equations of ideal hydrodynamics using a second order, finite volume, directionally un-split Godunov-type scheme, with an exact Riemann solver. The gravitational force is calculated with a split Tree-PM approach, where long-range forces are calculated from a particle-mesh method, and short-range forces are calculated with a hierarchical octree algorithm. Our galaxy formation model is based on the inclusion of several additional ... | 06:41 |
| kanzure | ... astrophysical processes: Gas cooling and photo-ionization ... Star formation and ISM model ... Stellar evolution .. Stellar feedback .. Black holes and SMBH feedback" | 06:41 |
| kanzure | simulation details http://www.illustris-project.org/about/#astronomers-three | 06:41 |
| kanzure | results http://www.illustris-project.org/about/#astronomers-four | 06:41 |
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| heath | #xmas http://dangerousprototypes.com/docs/Bus_Pirate | 06:51 |
| heath | .title | 06:51 |
| yoleaux | Bus Pirate - DP | 06:51 |
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| kanzure | wik Dr. Zeus Inc. | 07:13 |
| kanzure | .wik Dr. Zeus Inc. | 07:13 |
| yoleaux | "Dr. Zeus Inc., also known simply as The Company, is a fictional entity in a series of time travel science fiction stories by Kage Baker." — http://en.wikipedia.org/wiki/Dr._Zeus_Inc. | 07:13 |
| kanzure | "According to the stories, Dr. Zeus operates from the 24th century, using technologies of time travel and immortality to exploit the past for commercial gain. The immortality technology is limited to taking young children and turning them into cyborgs. The time travel technology only allows journeys into the past, and returns to the present. No artifacts or people can be brought forward from their own times. In addition, the technology ... | 07:13 |
| kanzure | ... is expensive and dangerous for normal humans to use." | 07:13 |
| kanzure | "To carry out its mission, Dr. Zeus sends its employees far into human prehistory, where they take children from Neanderthal and modern human families and give them the immortality treatment. These individuals are then promised a bright future in the 24th century, in exchange for working for the Company till then. Their job is to preserve cultural artifacts, valuable plants, and endangered species, hiding them in safe places till the ... | 07:14 |
| kanzure | ... Company can 'recover' them in the future. The cyborgs will get to the 24th century the old-fashioned way, by living through the intervening millennia. Along the way they can create others to help them, using children who would otherwise die and not affect history. They are also provided with many recordings of future culture, entertainment, and a carefully edited view of history. Dr. Zeus alone knows everything that will happen up ... | 07:14 |
| kanzure | ... till the 24th century." | 07:14 |
| kanzure | pesky time travelers, messing everything up | 07:19 |
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| heath | http://dnastack.com/ga4gh/bob/map.html | 07:38 |
| heath | Global Alliance for Genomics and Health | 07:38 |
| heath | a search engine according to wired.uk | 07:38 |
| heath | http://genomicsandhealth.org/ | 07:38 |
| heath | by that group | 07:38 |
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| kanzure | narwh4l: http://diyhpl.us/wiki/dna/projects/#dna-synthesis | 07:57 |
| heath | https://www.ece.cmu.edu/~safari/pubs/kim-isca14.pdf | 08:07 |
| heath | "flipping bits in memory without accessing them" | 08:07 |
| narwh4l | that is awesome | 08:08 |
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| kanzure | "If bad people can use these technologies, we must use them even better." | 08:29 |
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| kanzure | 08:38 < gmaxwell> atgreen: As a random aside, have you seen tinyram? http://www.scipr-lab.org/doc/TinyRAM-spec-0.991.pdf it's a very simple risc designed to have a maximally small arithemetic circuit to verify that a transcript of execution was correct. Because the proof enviroments its targeted for are so slow they did care a fair bit about program size, and one of their papers has benchmarks vs x86/arm/avr | 08:40 |
| kanzure | https://eprint.iacr.org/2013/507.pdf (page 12) | 08:40 |
| heath | "Although Hwang deceived the world about being the first to create artificially cloned human embryos, he did contribute a major breakthrough to stem cell research by creating human embryos using parthenogenesis" | 08:40 |
| heath | https://en.wikipedia.org/wiki/Parthenogenesis#Humans | 08:40 |
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| nmz787 | kanzure: anything I mentioned last night about lasers was either for exposing photoresist or adding heat (the latter in regards to tDt) | 12:18 |
| kanzure | that was not obvious | 12:18 |
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| nmz787 | 00:16 < nmz787> I wonder if there is a thermo-regulatable tDt... chill the reactor then pulse with a laser/resistive-heater fast enough to incorporate only one unit | 12:20 |
| nmz787 | 00:16 < nmz787> I wonder if there is a thermo-regulatable tDt... chill the reactor then pulse with a laser/resistive-heater fast enough to incorporate only one unit | 12:20 |
| nmz787 | sorry for double-posting, irssi sent the return character from the copy buffer too I guess | 12:20 |
| eudoxia | i've gone back to xchat because i'm not as hardcore as i thought i was | 12:21 |
| kanzure | /set password oops | 12:22 |
| kanzure | nmz787: https://groups.google.com/d/msg/enzymaticsynthesis/3YEEv0OULo0/L5WGLyr6O1YJ | 12:22 |
| nmz787 | ugh, this stupid HTC phone really really pisses me off... I can't even see what the time of a missed call from yesterday was... they just don't offer that small piece of info | 12:26 |
| nmz787 | kanzure: I think he's just saying oligo library | 12:28 |
| nmz787 | but also a tRNA library too | 12:28 |
| nmz787 | so you didn't have to have all possible oligo combinations in your library | 12:29 |
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| kanzure | sounds like he means 21 * 21 to me | 13:25 |
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| kanzure | e.g. a tRNA library of amino acids, and then for each aa-tRNA a library of aa-tRNA-RNA (i forget which RNA it uses, tRNA? mRNA?) | 13:25 |
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| kanzure | .wik titin | 13:31 |
| yoleaux | "Titin /ˈtaɪtɪn/, also known as connectin, is a protein that, in humans, is encoded by the TTN gene. Titin is a giant protein, greater than 1 µm in length, that functions as a molecular spring which is responsible for the passive elasticity of muscle. It is composed of 244 individually folded protein domains connected by unstructured …" — http://en.wikipedia.org/wiki/Titin | 13:31 |
| kanzure | "It is composed of 244 individually folded protein domains connected by unstructured peptide sequences.[4] These domains unfold when the protein is stretched and refold when the tension is removed.[5]" | 13:32 |
| kanzure | "After myosin and actin, titin is the third most abundant protein in muscle and an adult human contains approximately 0.5 kg of titin.[8] With its length of ~27,000 to ~33,000 amino acids (depending on the splice isoform), titin is the largest known protein.[9] Furthermore, the gene for titin contains the largest number of exons (363) discovered in any single gene,[10] as well as the longest single exon (17,106 bp)." | 13:32 |
| kanzure | "Titin, a polypeptide chain protein that is greater than 1µm in length and 3-4 nm in width [2]." | 13:32 |
| kanzure | hmm. | 13:34 |
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| kanzure | so at 20 amino acids per second a ribosome would need 27.5 minutes to finish synthesizing one of those | 13:36 |
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| yashgaroth | I thought it was just you have a template of [AAAGGG] repeats like dan said, and 2x21 tRNAs - one set that sticks to AAA and the other to GGG, and you alternate adding them | 13:39 |
| kanzure | i have no idea which one cathal meant, but if 2x21 tRNAs works then that's cool | 13:40 |
| kanzure | i think i misunderstood how many molecules tRNA binds to | 13:41 |
| yashgaroth | I've no idea either, but if that was it, then yes it's a promising proposal | 13:41 |
| kanzure | i thought it was an amino acid plus a nucleotide/sequence/fragment | 13:41 |
| kanzure | but after watching molecular biology porn, i think not | 13:41 |
| kanzure | enzyme costs of the proposal are really unfortunate | 13:42 |
| yashgaroth | how so? you're reusing the ribosomes | 13:42 |
| kanzure | tRNAs? | 13:42 |
| kanzure | i suppose you could try to recycle tRNAs | 13:42 |
| yashgaroth | ah true but there's not much of a better option | 13:43 |
| kanzure | do you think this would really be helpful? | 13:44 |
| kanzure | i've been thinking about genomes for so long that i haven't thought much about protein synthesis as a viable first step | 13:44 |
| yashgaroth | other options won't be much less expensive, RNA is always a huge hassle to work with though | 13:45 |
| kanzure | can you transcribe DNA -> mRNA in the same pot you add your ribosomes into? | 13:45 |
| kanzure | i've never done a cell-free system before | 13:45 |
| kanzure | er, besides polymerase stuff | 13:46 |
| yashgaroth | in a normal cell-free system, sure, that's how it works in a cell anyway | 13:46 |
| kanzure | there would still be a ton of permutation/iteration in figuring out a dna polymerase that is usable for genome synthesis | 13:50 |
| yashgaroth | oh, yes | 13:51 |
| kanzure | i'm trying to get convinced by thinking about intermediate usefulness | 13:51 |
| kanzure | i don't have a list on the wiki like "proteins to make in the event that you are able to make massive proteins" | 13:53 |
| yashgaroth | you can make massive proteins in cells, I'm just saying the AAAGGG method was rather elegant | 13:54 |
| kanzure | you can't make massive proteins in cells unless (1) the protein is in that genome or (2) you pay a shitload of money to synthesize the genes | 13:56 |
| kanzure | er, by (1) i meant in the genome or in a plasmid | 13:56 |
| kanzure | or (3) you have the plasmid already, fine | 13:56 |
| QuadIngi | "so at 20 amino acids per second a ribosome would need 27.5 minutes" is that the actual AA production time for ribosomes, I thought it was something closer to 250 | 13:57 |
| yashgaroth | that's high, for titin it'd be mammalian so much slower | 13:57 |
| kanzure | my 20 amino acids/second number was definitely for a bacterial ribosome | 13:59 |
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| yashgaroth | wait did you mean 250 minutes, or 250 AAs per second | 13:59 |
| nmz787 | still seems more reasonable to try the tDt method if some reversible terminators can be found for a reasonable price... or some other more available method | 14:02 |
| kanzure | yashgaroth thinks the tdt method is silly | 14:02 |
| yashgaroth | just the way the cooper union people proposed it | 14:03 |
| nmz787 | what stood out as silly? | 14:05 |
| yashgaroth | the only advantages, as they admit, are fewer toxic chemicals, and "it only has two steps so it's faster" which is bullshit because thermal deprotection is gonna take forever | 14:06 |
| nmz787 | why would it take forever? | 14:06 |
| yashgaroth | t/2 of five minutes | 14:06 |
| nmz787 | the reaction time was known to be 5 mins for deprotection? | 14:06 |
| yashgaroth | five minutes for half of it | 14:06 |
| nmz787 | less toxic chems often means higher reaction vessel material compatibility | 14:07 |
| kanzure | http://en.wikipedia.org/wiki/Vault_%28organelle%29 | 14:07 |
| kanzure | "The vault or vault cytoplasmic ribonucleoprotein is a eukaryotic organelle whose function is not fully understood. Discovered and successfully isolated by cell biologist Nancy Kedersha and biochemist Leonard Rome of the UCLA School of Medicine in the 1980s, vaults are cytoplasmic organelles which under an electron microscope resemble the arches of a cathedral vault, with 39-fold symmetry.[1] They are present in many types of eukaryotic ... | 14:07 |
| kanzure | ... cells and appear to be highly conserved amongst eukaryotes.[2] Vaults become part of lipid rafts where they may play a role fighting pathogens.[3]" | 14:07 |
| nmz787 | microscale reactions will be much faster | 14:07 |
| yashgaroth | I'm seeing about 30-35 minutes for 99% deprotection, which I assume would be a minimum | 14:08 |
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| nmz787 | if you were using a micro/nano channel I would think the time would be seconds | 14:08 |
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| yashgaroth | doesn't magically make it faster | 14:09 |
| nmz787 | not magically of course | 14:09 |
| yashgaroth | chemical compatibility may be an advantage, I'm not sure how limiting that is | 14:09 |
| nmz787 | coating microfluidics with PTFE layers is more work and less easily available than silicones, silicons, or glass substrates | 14:10 |
| yashgaroth | oh also they're not gonna be able to just "find or engineer" a variant of tdt that's stable at 95C, but I suppose that's not a showstopper | 14:11 |
| nmz787 | are you getting the time numbers from the NEB paper? | 14:11 |
| yashgaroth | no, from their igem page | 14:11 |
| nmz787 | well you can just separate the deprotection from the enzyme reaction | 14:11 |
| nmz787 | really though, if you can detect with enough sensitivity, you can just control the flow to allow only a single nucleotide in | 14:13 |
| nmz787 | so terminators are obviated | 14:13 |
| yashgaroth | by single do you mean single molecule | 14:13 |
| nmz787 | one way to gain sensitivity is to decrease your reaction cross-section | 14:13 |
| nmz787 | yeah | 14:13 |
| kanzure | "Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein." er... okay. | 14:13 |
| * nmz787 thinks nanochannel and impedance spectroscopy with a dilute solution of nucleotides | 14:14 | |
| kanzure | so far nobody has demonstrated flow control of a single nucleotide --- | 14:14 |
| kanzure | er, ignore those --- | 14:14 |
| kanzure | so you should not assume that when imagining insanely hard engineering projects | 14:15 |
| nmz787 | I am tempted to buy that UV engraver, though I know the resolution will suck at less than probably 100 microns, but I need more help from fenn or others on the laser etcher design he started | 14:15 |
| kanzure | in general you should limit the number of insane components in insane engineering projects | 14:15 |
| nmz787 | ? | 14:15 |
| nmz787 | nanofluidics are proven and easy if you can fab nanoscale elements | 14:15 |
| nmz787 | you don't even need agarose or channel wall coating | 14:15 |
| nmz787 | to get nice separations | 14:16 |
| yashgaroth | using a dilute solution to assume you're flowing in one molecule every time sounds insane | 14:16 |
| nmz787 | no, you wouldn't assume | 14:16 |
| yashgaroth | so you're detecting each one? | 14:16 |
| nmz787 | that's what the impedance spectroscopy would be for, to verify | 14:16 |
| kanzure | i am not disputing the existence of nanofluidics, i am disputing the engineering strategy you are taking | 14:16 |
| nmz787 | idk seems easier than your enzyme coctail scheme | 14:16 |
| nmz787 | less custom shit other than a nanochannel | 14:17 |
| kanzure | a nanochannel is an incredibly specialized piece of equipment | 14:17 |
| nmz787 | off the shelf nucletides, maybe a kinase, and tDt... umm maybe some ligation downstream once you have reasonable sized oligos | 14:17 |
| nmz787 | not really | 14:18 |
| nmz787 | it's much easier to make a nanochannel than a cocktail of enzymes | 14:18 |
| nmz787 | and specialized functionalized organics | 14:18 |
| nmz787 | it is after all, just a channel | 14:18 |
| nmz787 | not a MEMS | 14:18 |
| nmz787 | or NEMS | 14:18 |
| kanzure | it is after all, just a dyson sphere | 14:18 |
| nmz787 | that is totally different | 14:19 |
| kanzure | both are engineering projects that, if you underestimate, will not be completable | 14:19 |
| nmz787 | ion mills are something achievable by DIY tech, I am not saying that it would be akin to a FIB (which can be steered) | 14:19 |
| nmz787 | and once a master exists, it can be copied by stamping | 14:22 |
| nmz787 | (and I just got called into FIB lab, so gotta go get ready) | 14:22 |
| kanzure | i think you are underestimating engineering failure modes | 14:22 |
| nmz787 | what failure mode are you considering? | 14:22 |
| yashgaroth | reliably detecting a single molecule | 14:23 |
| kanzure | every additional component you add to a project geometrically adds to the number of bugs and causes of bugs | 14:23 |
| nmz787 | idk the sequencing folks do it now with hotons | 14:23 |
| nmz787 | photoins | 14:23 |
| nmz787 | rught, which is why I want less components | 14:24 |
| QuadIngi | I meant 250 AA/second, but then I haven't learned this stuff yet | 14:24 |
| kanzure | yes but you chose "less components, but less engineered" | 14:24 |
| kanzure | do you know why people used vacuum tubes? | 14:25 |
| nmz787 | http://diyhpl.us/~nmz787/pdf/Nanofluidics_in_chemical_analysis_.pdf | 14:26 |
| nmz787 | because that is what was discovered first | 14:26 |
| kanzure | i already told you that i am not disputing the existence of nanofluidics, fuck you | 14:26 |
| kanzure | argh | 14:26 |
| nmz787 | fuck you assface | 14:26 |
| nmz787 | read the paper | 14:26 |
| nmz787 | it isn't about prood | 14:26 |
| nmz787 | proof | 14:26 |
| nmz787 | gah | 14:26 |
| nmz787 | you are almost as bad as my gf when it comes to getting loud quickly | 14:27 |
| kanzure | my knowledge of nanofluidics has exactly zero bearing on this conversation | 14:27 |
| nmz787 | obviously | 14:27 |
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| kanzure | planar microelectronics could have easily been known before vacuum tubes, you just don't know, they chose vacuum tubes because they knew they worked | 14:28 |
| kanzure | now you can tell me you "know" nanofluidics works | 14:28 |
| kanzure | there's a big difference between the different nasa technical readiness levels | 14:29 |
| kanzure | http://esto.nasa.gov/files/trl_definitions.pdf | 14:29 |
| kanzure | in general you should maximize the number of higher technical readiness level components in a project, and minimize the number of lower numbered readiness components | 14:29 |
| nmz787 | they didn't know how to grow single crystals when they invented vacuum tubes | 14:29 |
| nmz787 | all I'm saying is your ideas currently seem vastly more complex, less available, more expensive, more closed-source | 14:30 |
| kanzure | i haven't mentioned licensing at all, so i'm not sure why you think it is closed source | 14:31 |
| kanzure | ribosomes and tRNAs are widely available | 14:31 |
| nmz787 | because specialized chemicals have to be made by some specialized process, which is likely not free | 14:32 |
| kanzure | probability in engineering projects works by multiplying the probability of solving each problem, if the typical probability of a team successfully capturing a polymerase in a nanofluidic channel is 0.10 then you must include that number when multiplying your theoretical area components together | 14:32 |
| nmz787 | I never said anything about capturing a polymerase in a nanofluidic channel | 14:34 |
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| kanzure | sum of ((probability of capturing a polymerase) * (probability of implementing a working single nucleotide detector) * (probability of gate 1 working) * (probability of gate N working) * (probability of layer alignment working)) etc etc for each thing | 14:34 |
| nmz787 | please stop interpreting things so far and then blaming me for your wrong interpretations | 14:34 |
| kanzure | you have known for at least two years now that i would interpret nanofluidic ideas to be referring to polymerase capture, that's what you and i were talking about | 14:34 |
| kanzure | especially when you mention single nucleotide capture and transfer | 14:35 |
| nmz787 | I specifically mentioned cross-section of a channel to increase detection sensitivity | 14:35 |
| kanzure | anyway, polymerase capture is just one example | 14:35 |
| kanzure | generally, the probability of project success decreases as you increase the number of low-technological-readiness-level components, even if you are confident that, given enough time, you can debug each and every component | 14:36 |
| nmz787 | right, which is why specialized chems and enzymes are not a good idea | 14:36 |
| nmz787 | a tiny-ass channel is not hard to thingk about of find decades of research on | 14:37 |
| nmz787 | or single-molecule detecion | 14:37 |
| nmz787 | especially regarding nucleotides or polynucleotides | 14:37 |
| kanzure | no, enzymatic protein synthesis is a bad idea because you don't know if ribosomes can survive washes or are okay waiting for very long periods, but otherwise all of the other components are fairly well known (hell, there's even fucking cell-free protein synthesis kits on the market) | 14:37 |
| nmz787 | I am not saying this is the only way | 14:37 |
| nmz787 | I am just saying that more chems that are hard to get is not a good idea | 14:38 |
| nmz787 | more reactions to think about is not a good idea. | 14:38 |
| yashgaroth | what're the chemicals? | 14:38 |
| nmz787 | more convuluted purification schemes is not a good idea. | 14:38 |
| nmz787 | the cell-free protein kits require mRNA as input | 14:38 |
| kanzure | yes, it's true that you will need to load up some known mRNA strand, and yes it sucks storing mRNA, but mRNA synthesis is known and only has to be done "once" (if i store it right after doing it once) | 14:39 |
| nmz787 | then how do you permute? | 14:40 |
| kanzure | say again? | 14:40 |
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| nmz787 | if you have some mRNA, where did you get it? | 14:41 |
| nmz787 | the point is to make mRNA/protein on-demand, right? | 14:41 |
| kanzure | only proteins on demand, not mRNA | 14:42 |
| nmz787 | ok, but cell-free protein synthesis needs mRNA. So where does the permutation come into play here? | 14:42 |
| kanzure | originally my proposal required some bullshit engineering of a ribosome | 14:42 |
| kanzure | cathal made a proposal that eliminated that requirement | 14:42 |
| yashgaroth | we talking the template strand mRNA? that's a generic sequence | 14:42 |
| kanzure | his suggestion was to synthesize an mRNA strand once, and just copy/cone that | 14:43 |
| nmz787 | right, but then you need a source of tRNAs too | 14:43 |
| kanzure | tRNAs are just enzymes or something | 14:43 |
| kanzure | they might be ribosomethings | 14:43 |
| nmz787 | which either means buying then (likely not cheap) or making/regenerating them (more steps, more reactions, more purifications) | 14:43 |
| kanzure | ah, enzyme | 14:43 |
| kanzure | *clone | 14:45 |
| nmz787 | so now instead of flowing in single nucleotides to tDt, you're flowing in single tRNAs to a ribosome... and hope that choking is OK and that the protein doesn't fall out | 14:45 |
| kanzure | nope not single tRNAs | 14:45 |
| nmz787 | ? | 14:45 |
| nmz787 | if the mRNA is generic, then it is just road for the ribosome to walk down | 14:45 |
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| nmz787 | the tRNAs are what define the AA seq | 14:45 |
| kanzure | i think you only need single tRNA types, but not control over single tRNA molecules | 14:46 |
| nmz787 | then you would always get the same AA out for the same generic mRNA | 14:46 |
| kanzure | there are tRNAses in the literature that have been modified for this purpose | 14:47 |
| kanzure | this is the only questionable, unknown element in the entire scheme | 14:47 |
| kanzure | other than ribosome survival during wash | 14:47 |
| nmz787 | CV==cyclic voltammetry -- "In a typical CV measurement of guanine or adenine, the concentration of the bases is in the range of 10-100 µM [94, 97]. For a single-molecule confined in a nanochannel described above, due to the small volume of the channel, the average concentration of the bases is 4.4 mM, orders of magnitude higher than the concentration necessary for the conventional bulk measurements." | 14:47 |
| kanzure | and growing-strand protein survival during wash | 14:47 |
| nmz787 | https://www.princeton.edu/physics/graduate-program/theses/theses-from-2008/C.Tungthesis.pdf | 14:47 |
| kanzure | and whether ribosomes can resume after waiting multiple minutes per tRNA | 14:48 |
| kanzure | yashgaroth: how long are the steps in cell-free kits? | 14:48 |
| kanzure | oh wait, ribosome goes as fast as it can | 14:48 |
| nmz787 | "[94] S. C. Brooks and M. M. Richter, “Determination of DNA bases using electrochemistry: A discovery-based experiment,” Chem. Educators, vol. 7, pp. 284– 287, 2002" | 14:48 |
| yashgaroth | mhm | 14:48 |
| nmz787 | "[97] K. Wu, J. Fei, W. Bai, and S. Hu, “Direct electrochemistry of DNA, guanine and adenine at a nanostructured film-modified electrode,” Anal. Bioanal. Chem., vol. 376, pp. 205–209, 2003." | 14:48 |
| yashgaroth | that is a concern, would be interesting to do some directed evolution to get a more tolerant ribosome | 14:48 |
| kanzure | you are arguing science to me, and i was arguing engineering | 14:49 |
| nmz787 | directed evolution is both | 14:49 |
| nmz787 | sounds like I'm one-up | 14:49 |
| kanzure | directed evolution is frosting i think | 14:49 |
| yashgaroth | I just said it'd be interesting jeez | 14:49 |
| kanzure | my comment about arguing engineering was intended for nmz787 | 14:50 |
| yashgaroth | as was mine just now heh | 14:50 |
| kanzure | nmz787: in general, many academic papers are like "we achieved these results, under these 1 million conditions that are totally impossible to replicate without like a few million dollars or something" | 14:51 |
| kanzure | so combining the number of different papers you need to solve a problem just multiplies these frustrations | 14:51 |
| nmz787 | sure, but voltammetry has been around for like 100 years | 14:51 |
| nmz787 | but it's sure easier than a shitload more chems/enzymes/steps/etc | 14:51 |
| yashgaroth | but all 42 tRNAs are produced the same way | 14:52 |
| nmz787 | http://diyhpl.us/~nmz787/pdf/Direct_electrochemistry_of_DNA_guanine_and_adenine_at_a_nanostructured_film-modified_electrode.pdf | 14:52 |
| nmz787 | paperbot: http://link.springer.com/article/10.1007%2Fs00897020595a | 14:53 |
| kanzure | yashgaroth: honestly i might prefer a totally-directed-evolution-only method of trying to achieve human control of enzymatic synthesis. the problem is that directed evolution is only really good at things that are already working and you want more of.... | 14:54 |
| kanzure | and also you need weird things like tagged display of mRNA/sDNA/dsDNA on attached results so that you can identify/promote beneficial sequences | 14:55 |
| kanzure | which is v. problematic | 14:55 |
| nmz787 | hmm, do doi numbers get deprecated? | 14:55 |
| nmz787 | the one mentioned here doesn't work https://web.archive.org/web/20101212114751/http://chemeducator.org/sbibs/s0007005/spapers/750284mr.htm | 14:55 |
| nmz787 | http://dx.doi.org/10.1007/s0089702595b | 14:55 |
| kanzure | ask dingo when he shows up again | 14:56 |
| kanzure | .to dingo are doi numbers ever deprecated? | 14:56 |
| yoleaux | kanzure: I'll pass your message to dingo. | 14:56 |
| kanzure | yashgaroth: how about this.... consider a phosphoramidite oligonucleotide synthesis scheme, where you also include a bunch of random polymerase enzymes, cofactor enzymes, just lots of random shit. then, you do stepwise chemical synthesis. later, you pcr the results, then turn those back into enzymes and start the process over again | 14:58 |
| kanzure | er, this is assuming you are doing some sort of 1024x1024 synthesis method maybe | 14:58 |
| kanzure | and er, you would want to proteins that are better at helping the synthesis process to contribute to preserving their own sequence | 14:58 |
| kanzure | i guess you would have to sequence each time before putting enzymes into separate reaction chambers :( | 14:59 |
| yashgaroth | yeah it'd be a big hassle, I'm hoping it's something easy like doing a hundred random mutants which cripple the ribosome just enough to keep it from dissociating | 15:00 |
| kanzure | (because you have to photo-optically signal the correct sequence for each chamber) | 15:00 |
| kanzure | oh, i wasn't talking about ribosome directed evolution stuff, but i see what you mean | 15:00 |
| kanzure | i just meant "using random enzymes in phosphoramidite reactions to see which enzymes might help improve the coupling efficiencies" | 15:00 |
| kanzure | "and yield" | 15:01 |
| yashgaroth | you mean in a traditional chemical dna synthesis? | 15:01 |
| kanzure | yes | 15:01 |
| yashgaroth | wouldn't they get all fucked up on chemicals | 15:01 |
| kanzure | apparently tdt didn't in that igem project | 15:01 |
| kanzure | so there's one example haha | 15:01 |
| kanzure | oh wait, did it denature? | 15:01 |
| yashgaroth | oh if you're doing it like tdt sure, though that one I presume denatured at every 95C step | 15:02 |
| kanzure | ouch | 15:03 |
| kanzure | yeah that wont work for directed evolution :) | 15:03 |
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| nmz787 | but you could just move your DNA elsewhere to heat | 15:03 |
| nmz787 | :/ | 15:03 |
| kanzure | i think you mean move your tdt | 15:04 |
| nmz787 | that is the problem with all this bulk-reaction stuff | 15:04 |
| nmz787 | no | 15:04 |
| nmz787 | I mean move the DNA | 15:04 |
| nmz787 | the tDt would be physically impeded more easily than the DNA | 15:04 |
| nmz787 | and you can move DNA with electrophoresis reliably | 15:04 |
| yashgaroth | but moving it far enough away that heating it to 95C for half an hour is kept isolated from the tdt? | 15:07 |
| yashgaroth | might be easier to just add fresh enzyme every time | 15:07 |
| kanzure | directe evolution would be the easiest answer, realy | 15:08 |
| kanzure | *really | 15:08 |
| nmz787 | what needed denatured anyway?> | 15:08 |
| kanzure | *directed | 15:08 |
| nmz787 | that was assuming you used some unobtainable reversible terminators | 15:09 |
| nmz787 | right? | 15:09 |
| yashgaroth | oh I meant mutating the ribosomes so they wouldn't spit out the protein while waiting for the next tRNA to wash in | 15:09 |
| kanzure | my comment about tdt was about whether or not tdt gets denatured in that igem team's method | 15:09 |
| kanzure | i don't know, i should look, but i wont | 15:10 |
| yashgaroth | it hella does | 15:10 |
| kanzure | ah | 15:10 |
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| nmz787 | hmm, I wonder if you had a nanopore (not an enzyme nanopore, just a pinhole) with a stable membrane over it, forming a diffusion barrier... if a pulse from electrphoresis plates could bring a single molecule through, and still allow the membrane to reseal | 15:12 |
| kanzure | i think the problem with directed evolution for human-controlled synthesis (of dna polymerase) is that you would need really really really cheap sequencing, because you *must* synthesize the mutated sequence while the mutated enzymes are helping.... but then do this like a million or a billion times (in parallel).. | 15:12 |
| kanzure | *control of dna polymerase | 15:12 |
| nmz787 | well, or perfect synthesis | 15:12 |
| nmz787 | how often does RAM get checked after being written? | 15:13 |
| nmz787 | unless it is ECC or memtest | 15:13 |
| kanzure | oh right, you can't do that with dna synthesis right now because oligos long enough to code for polymerase are way longer than the 100-200 bp you can reliably make | 15:13 |
| kanzure | that's even more unfortunate | 15:14 |
| kanzure | another nail in the directed evolution megacoffin for this problem | 15:14 |
| nmz787 | but once you get to that range voltammetry or traditional dyes can tell your if you have succesful lligation products of the constituent 100bp oligos | 15:14 |
| nmz787 | so stitching oligos is not a problem I think, as long as you're only dealing with single molecules at a time | 15:15 |
| nmz787 | or very low number population of molecules | 15:15 |
| nmz787 | since you can just filter the incorrect ones on the spot | 15:15 |
| kanzure | um, if you already have your magical single molecule device you don't need to do directed evolution of a human-controllable polymerase | 15:15 |
| nmz787 | again using electrophoresis | 15:15 |
| nmz787 | well you would still want to do directed evolution on other things | 15:16 |
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| kanzure | other things have much easier directed evolution techniques available to them | 15:16 |
| kanzure | such as in vivo cellular stuff | 15:16 |
| nmz787 | idk, as I recall it can be very very hard to setup 'reward/success function' for intermediates of a say an interesting operon that made some interesting molecule | 15:17 |
| nmz787 | like how do you setup feedback for: algae -- make butanol them pump it out immediately | 15:18 |
| nmz787 | in-silico permutations may be more appropriate | 15:19 |
| nmz787 | because we know a bit about photophysics in the chromophores | 15:19 |
| nmz787 | etc | 15:19 |
| nmz787 | and we know that sometimes fusion enzymes setup a 'pipeline' for operations | 15:20 |
| kanzure | those nonribosomal peptidyl synthetases look particularly insane | 15:20 |
| kanzure | http://www.pnas.org/content/101/44/15585/F1.large.jpg | 15:21 |
| nmz787 | I am gonna post to the dorkbot pdx list seeking help on the laser etcher | 15:21 |
| nmz787 | how does that sound? | 15:21 |
| kanzure | all of those "modules" in that image are in a giant tunnel in a giant protein | 15:21 |
| kanzure | uh, whatever, free country? | 15:21 |
| kanzure | i don't know what you're asking me | 15:21 |
| nmz787 | since I didn't get any responses here re that and the aliexpress link... well someone did say it looked cool | 15:21 |
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| juri_ | kanzure: you here yet? | 15:23 |
| nmz787 | I can't remember why fenn didn't like this style http://www.aliexpress.com/item/3040-CNC-router-milling-machine-mechanical-kit-ball-screw/1904262918.html | 15:23 |
| kanzure | juri_: what? | 15:23 |
| kanzure | i'm not attending 37c3 if that's what you mean | 15:24 |
| juri_ | sorry. wrong channel. ;) | 15:24 |
| nmz787 | this one is literally two CDROM trays with some plastic, at least it uses open-source hardware (easydriver, arduino, grbl) ... | 15:26 |
| kanzure | nmz787: btw, for the record, i still think microfluidics is a good idea for the planar photogated projector grid oligo synthesis | 15:26 |
| nmz787 | ... http://www.ebay.com/itm/Mini-Laser-200-250mW-Engraving-Machine-DIY-Carving-Logo-Picture-Marking-Printer/261591526091?_trksid=p2047675.c100010.m2109&_trkparms=aid%3D555012%26algo%3DPW.MBE%26ao%3D1%26asc%3D20131231084308%26meid%3Dbd34a3a2adef4c4296471dc22ffa982c%26pid%3D100010%26prg%3D20131231084308%26rk%3D5%26rkt%3D24%26mehot%3Dpp%26sd%3D221644113544&autorefresh=true | 15:26 |
| nmz787 | the tdt single molecule stuff would be cheaper and easy to try, and less harsh chems is a super duper + | 15:27 |
| nmz787 | like 4 vials of G C T A is like $150 or $200 | 15:29 |
| nmz787 | the synth chems alone were around $1000 or 1300 | 15:29 |
| kanzure | honestly the most frustrating part about the chemicals for that oligo synthesizer is the expiration of each chemical, so things have to be ordered all at once and they damn well better arrive all at once | 15:31 |
| kanzure | maybe those expirations are just lies though | 15:32 |
| kanzure | hard to tell if that's a consumer-bullshit thing, or an actual expiration | 15:32 |
| nmz787 | it's that water hurts the process | 15:35 |
| nmz787 | it is a fake 3'OH | 15:35 |
| nmz787 | at least that's a big one | 15:36 |
| kanzure | er, what water? | 15:36 |
| kanzure | i thought they arrive as powders | 15:36 |
| nmz787 | in the air | 15:36 |
| kanzure | air flushing back up through the tubes? | 15:37 |
| nmz787 | water can seep through plastic bottles, or cracks in a bottle seal | 15:37 |
| nmz787 | PDMS is permeable | 15:37 |
| nmz787 | for example | 15:37 |
| kanzure | enough to make the entire dry powder useless within 3-4 weeks? | 15:37 |
| nmz787 | i don't think that long | 15:38 |
| kanzure | even shorter? | 15:38 |
| nmz787 | but it would be a decay effect | 15:38 |
| kanzure | what sorta system did they make, it just dies the moment you look at it | 15:38 |
| nmz787 | your extension products would terminate more often | 15:38 |
| nmz787 | err, have a deletion error | 15:39 |
| nmz787 | but that is contamination | 15:39 |
| nmz787 | not the chem going bad | 15:39 |
| kanzure | yes it sounds like contamination is the only possible cause here | 15:39 |
| kanzure | otherwise storing everything unused for months should be okay | 15:39 |
| nmz787 | I can't remember though myself | 15:40 |
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| nmz787 | everything got dissolved when you wanted to use it though | 15:40 |
| nmz787 | so after that is when things will start souring more quickly | 15:40 |
| nmz787 | but some of them were also sure-seal bottles | 15:41 |
| nmz787 | which makes handling harder too | 15:41 |
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| nmz787 | which would be for water and also possibly for oxygen | 15:42 |
| nmz787 | s/water/moisture/ | 15:42 |
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| kanzure | "There have been bits of research results on the transformation from electrical signals to biological signals, for example, the phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor in Ciona intestinalis. However, the opposite direction of transformation is almost a blank space, especially in microbes." | 15:50 |
| kanzure | paperbot: http://www.nature.com/nature/journal/v435/n7046/abs/nature03650.html | 15:50 |
| kanzure | .title | 15:50 |
| paperbot | http://libgen.org/scimag/get.php?doi=10.1038%2Fnature03650 | 15:51 |
| yoleaux | kanzure: Sorry, that doesn't appear to be an HTML page. | 15:51 |
| kanzure | "Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor" | 15:51 |
| kanzure | "Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1–S4 segments of voltage-gated channels and a cytoplasmic domain similar to phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing phosphatase (Ci-VSP), displays channel-like 'gating' currents and directly translates changes in membrane potential into the ... | 15:52 |
| kanzure | ... turnover of phosphoinositides. The activity of the phosphoinositide phosphatase in Ci-VSP is tuned within a physiological range of membrane potential." | 15:52 |
| kanzure | "Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized." | 15:52 |
| kanzure | "A hot-sensing cold receptor: C-terminal domain determines thermosensation in transient receptor potential channels" | 15:53 |
| kanzure | "Engineering and characterization of an enhanced fluorescent protein voltage sensor" http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0000440 | 15:55 |
| kanzure | oh, that is membrane-bound too | 15:56 |
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| nmz787 | .title http://www.ncbi.nlm.nih.gov/pubmed/23965997 | 15:58 |
| yoleaux | Structure of the type IVa major pilin from the electrically conduct... - PubMed - NCBI | 15:58 |
| nmz787 | Structure of the type IVa major pilin from the electrically conductive bacterial nanowires of Geobacter sulfurreducens. | 15:58 |
| kanzure | oh. | 15:59 |
| nmz787 | Fig 5 C, schematic diagram showing the progression of the aromatic clusters up the pilus structure. The aromatic band is colored blue, and the aromatic devoid band is colored yellow. | 16:00 |
| nmz787 | huh, kinda interesting | 16:00 |
| nmz787 | http://diyhpl.us/~nmz787/pdf/Type_IV_Pilin_Proteins_Versatile_Molecular_Modules.pdf | 16:03 |
| nmz787 | paperbot: http://www.nature.com/nature/journal/v435/n7045/full/nature03661.html | 16:03 |
| paperbot | http://libgen.org/scimag/get.php?doi=10.1038%2Fnature03661 | 16:03 |
| kanzure | this one is okay, | 16:03 |
| kanzure | http://femsre.oxfordjournals.org/content/early/2014/11/27/femsre.fuu001 | 16:03 |
| paperbot | http://libgen.org/scimag/get.php?doi=10.1093%2Ffemsre%2Ffuu001 | 16:04 |
| kanzure | also mentions type iii secretion systems hehe | 16:04 |
| nmz787 | I didn't find any decent hits with: "PilA" reporter electrical | 16:04 |
| kanzure | "As for competence pseudopili, T2SS pseudopili have never been directly visualized, probably because they are too short, barely spanning the periplasmic space (Douzi, Filloux and Voulhoux 2012; Nivaskumar and Francetic 2014). However, a key piece of evidence for their existence is the fact that surface-exposed filaments similar to Tfp, named ‘hyper-pseudopili’, can be seen when T2SS-harbouring bacteria are genetically engineered to ... | 16:05 |
| kanzure | ... overexpress the major pseudopilin (Sauvonnet et al., 2000; Durand et al., 2003; Vignon et al., 2003). T2SS-exported proteins, often enzymes, play important roles in lifestyles of the secreting bacteria by providing them with essential nutrients or by having toxic effects on host cells in pathogens (Nivaskumar and Francetic 2014). " | 16:05 |
| nmz787 | .title http://www.google.com/patents/US20140239237 | 16:06 |
| yoleaux | Patent US20140239237 - Microbial nanowires and methods of making and using - Google Patents | 16:06 |
| kanzure | "Direct electrochemistry of redox enzymes as a tool for mechanistic studies" http://pubs.acs.org/doi/pdf/10.1021/cr0680742 | 16:10 |
| kanzure | "Nanoporous gold film encapsulating cytochrome c for the fabrication of a H2O2 biosensor" | 16:13 |
| nmz787 | "Although it has been proposed that conductivity might result from electrons ‘hopping’ between cytochromes attached to Geobacter Tfp (Boesen and Nielsen 2013), filaments are likely to have intrinsic metal-like electron-conductive properties through stacking of aromatic residues of the major pilin PilA. Accordingly, filaments composed of pilA mutants lacking these residues have reduced conductive properties (Vargas et al., 2013)." | 16:13 |
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| kanzure | hemophiliacs that take injections of FVIII sometimes develop immunity to FVIII, so these people are trying personalized FVIII based on gene from patient :/ http://www.fda.gov/BiologicsBloodVaccines/ScienceResearch/BiologicsResearchAreas/ucm246804.htm | 17:00 |
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| kanzure | 17:36 < gmaxwell> apparently the tor controller port can now create hidden services: https://stem.torproject.org/tutorials/over_the_river.html | 17:56 |
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| kanzure | .title https://groups.google.com/forum/#!topic/diybio/Vq_OJ4RgR9U | 18:01 |
| yoleaux | Google Groups | 18:01 |
| kanzure | oh good i quoted the content | 18:02 |
| kanzure | https://groups.google.com/d/msg/diybio/Vq_OJ4RgR9U/g3mu_ID7mrkJ | 18:02 |
| kanzure | ParahSailin: ^ | 18:02 |
| kanzure | huh i had reasonable things to say back then? https://groups.google.com/d/msg/diybio/GZAhcDBYsws/aoxblbq6a-wJ | 18:05 |
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| kanzure | 21:08 < gmaxwell> wumpus: well wrt impossible: this is the amount of work at the current hashrate that it would take to replace the whole chain: http://bitcoin.sipa.be/powdays-50k.png in days | 21:09 |
| maaku | it's been trending up, thankfully | 21:17 |
| maaku | it was scary low in the beginning of the year | 21:17 |
| kanzure | geodesic dome connector https://medium.com/dome-kit/slow-flexible-cheap-5598ca91fb38 | 21:27 |
| kanzure | hardware acceeration of key-value stores http://zhehaomao.com/papers/memcached-fpga-accel.pdf | 21:28 |
| kanzure | octopus pluralization http://freethoughtblogs.com/pharyngula/files/2012/03/pluralizingoctopus.jpg | 21:32 |
| kanzure | octopus grammar translation https://www.youtube.com/watch?v=KMM4XYteqWI | 21:35 |
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| kanzure | http://freethoughtblogs.com/pharyngula/2014/08/01/friday-cephalopod-theyre-forming-tribes/ "Panamanian biologist Aradio Rodaniche first reported the Pacific striped octopus in 1991 off the coast of Nicaragua, noting its strange behavior—living in groups of possibly up to 40, laying multiple egg clutches, and mating face-to-face and sucker-to-sucker. Most other octopus species, for instance, come together only to mate." | 21:39 |
| kanzure | http://news.nationalgeographic.com/news/2014/07/140728-social-octopuses-animals-oceans-science-mating/ | 21:40 |
| kanzure | http://ib.berkeley.edu/people/faculty/caldwellr | 21:42 |
| superkuh | "This project was unveiled on April 1, 2012." | 21:43 |
| kanzure | aww. | 21:43 |
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| nmz787 | "The focal length of an electrostatic lens is dependent on the energy spread of the ions. It is analogous to a simple glass lens focusing different wavelengths of light at different points." | 23:22 |
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