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| chris_99 | dumb question, is it ok to use food grade agar for lab stuff, or.. should i be buying a different grade | 09:20 |
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| chris_99 | (for the plant tissue culturing) | 09:20 |
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| nmz787 | chris_99: you should be fine, but ask sebastian cocioba | 11:24 |
| chris_99 | cool, i'm not sure who that is, is he on this channel? | 11:25 |
| nmz787 | wait, now I am confused as to whether you're supposed to have a 4th of July party on the 3rd (so you're partying when the clock rolls over) or if you actually should celebrate on the 4th | 11:28 |
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| nmz787 | chris_99: I don't think so, I can find his email | 11:28 |
| nmz787 | chris_99 scocioba@gmail.com | 11:28 |
| chris_99 | cheers | 11:29 |
| chris_99 | is he on the DIYbio list or something | 11:29 |
| nmz787 | just say I sent you, or leave that out and make something up about an auto-mailer bot that has been spamming ALL email addresses serially since 1999, asking about this same topic | 11:30 |
| nmz787 | yeah DIYbio google group | 11:30 |
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| chris_99 | ah heh cheers | 11:30 |
| nmz787 | how much should I offer someone on craigslist for a fume hood that they want $350 for... but has been listed for at least a month or two | 11:37 |
| chris_99 | would $250 be too much of a difference | 11:38 |
| nmz787 | it would be an improvement on my side of the deal, for sure... I am just wondering if that is still too high or ont | 11:39 |
| nmz787 | not | 11:39 |
| nmz787 | my value system is all messed up by cheap chinese goods | 11:39 |
| nmz787 | I imagine the least the person would get is as scrap metal weight... assuming they never sell it | 11:40 |
| nmz787 | chris_99: have you ever worked with micropython? | 11:40 |
| chris_99 | nope, i've very briefly played with elua on an esp8266 | 11:41 |
| chris_99 | though | 11:41 |
| nmz787 | ah, I just got a STM Nucleo F401 flashed with micropython a few nights ago and am playing with it a bit | 11:48 |
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| nmz787 | goal is to just do some GPIO toggling, which should hook up to some coil drivers to make high-voltage pulses for shoving DNA into bacteria | 11:49 |
| chris_99 | ah neat the electroporation thing? | 11:50 |
| nmz787 | yeah | 11:52 |
| nmz787 | so far my one test for dimming an LED wasn't successful | 11:52 |
| xentrac | too slow? | 11:54 |
| nmz787 | that is my thought | 11:54 |
| chris_99 | how are you gonna generate the pulses | 11:55 |
| nmz787 | there is some LED.intensity method... but it just made the LED full brightness for all 0-255, except 0 which was off, and 1 which was slightly dimmer than full bright | 11:55 |
| nmz787 | I think I might just need to use inline-ASM | 11:55 |
| nmz787 | another person is doing the bulk of the current R&D | 11:55 |
| nmz787 | so he is working on coils and such | 11:55 |
| xentrac | Arduino has similar behavior if you try to PWM a pin that doesn't have PWM | 12:03 |
| xentrac | it also kind of sounds like the kind of behavior you'd get out of an AVR PWM driver if the limit register was set wrong | 12:06 |
| xentrac | I don't know anything about STM chips but it wouldn't surprise me if they worked the same way | 12:07 |
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| nmz787 | xentrac: nah I think I'd have had success bit-banging an arduino GPIO | 12:08 |
| chris_99 | have you checked the output | 12:09 |
| nmz787 | you can do MHZ if I recall, in a busy loop | 12:09 |
| chris_99 | from the pin, with a scope or something | 12:09 |
| nmz787 | nope, I was in a motel room on the bed after my gf turned off the lights | 12:09 |
| chris_99 | ah | 12:09 |
| nmz787 | was using the LED as indicator | 12:09 |
| xentrac | sometimes in situations like that I've used an 8Ω speaker as an indicator | 12:15 |
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| kanzure | i thought made-in-space was doing asteroid things. but apparently most of what they are doing is 3d printing on the international space station. :\ | 12:33 |
| kanzure | nmz787: offer some amount lower, but also offer to pick it up yourself. | 12:35 |
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| CaptHindsight | 3d printing on the international space station? FDM, SLS, SLA, inkjet, DMLS? | 12:40 |
| chris_99 | http://www.nasa.gov/content/open-for-business-3-d-printer-creates-first-object-in-space-on-international-space-station sounds like it's FDM reading that? | 12:42 |
| chris_99 | although that's an old article | 12:42 |
| xentrac | it occurred to me the other day that FDM might work better on a vertical surface than a horizontal one | 12:46 |
| xentrac | you could eliminate the droop problem from bridges | 12:47 |
| kanzure | CaptHindsight: i think they are doing plastic extrusion heating things | 12:47 |
| kanzure | although i didn't ask what type of printing | 12:47 |
| kanzure | i doubt it is inkjet.. | 12:47 |
| xentrac | I mean you would still have droop but it wouldn't cause the deposited plastic to droop away from the bridging plane, but rather in it | 12:48 |
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| kanzure | re: controlled polymerase, perhaaps it would be good to start with selection experiments for really really slow polymerase. this will be trivial to do, something like an emulsion in vitro experiment with a billion trillion bubbles or something, and have each polymerase copy its own DNA. etc. and to select for slowness, you could probably segregate by mass because the slower ones will have lower mass at first. after completing this, you ... | 12:58 |
| kanzure | ... would then select for the polymerase that are very slow but where you can cause them to move forward and incorporate a single nucleotide perhaps by electrical stimulation or some other stimulation method. once you have a fully controlled polymerase, you can then fix the slowness by selecting for speed. | 12:58 |
| kanzure | and selecting for speed is easier i think: give it 5 hours to replicate itself, select everything that worked, then next round give 4.8 hours, etc. over time you have something that only takes 1 hour or 10 minutes. (although i think there's probably some physical limitations to polymerase speed) | 13:02 |
| kanzure | and it would be helpful to increase the physical size of polymerase, either for physical handling or for increasing the number of amino acids and residues that can be used for probing for susceptibility to external control. | 13:05 |
| kanzure | hm wait, mass wont work for selecting for slowness-- it's just a physical restructuring of the same mass, bubble will still have the same mass before and after. so that wont work. | 13:06 |
| kanzure | i guess you could look at what makes polymerase fast (by selecting for speed), and then just ensure you don't try anything similar to those changes :\ | 13:06 |
| nmz787 | seems like you'd need to use sequencing or at least a nice capillary electrophoresis to discriminate length | 13:08 |
| kanzure | temporal length, not physical length | 13:08 |
| nmz787 | then "run for time" and then "seqeunce" then "select for complete replicons" | 13:08 |
| nmz787 | yeah | 13:09 |
| nmz787 | but they are related... you want full physical lenght in less temporal | 13:09 |
| kanzure | "select for complete rpelicons" is easy in polymerase selection experiments-- they are self-replicating machines. you attach the dna to each polymerase and you also do some in vitro protein ribosome stuff. inside each emulsion bubble or something. | 13:09 |
| kanzure | i forget all the setup details | 13:10 |
| kanzure | nmz787: what was your synthesis idea? | 13:10 |
| kanzure | from the other day | 13:10 |
| kanzure | i think it might be possible to have conformational shape changes inside of the polymerase molecule that block nucloetide incorporation. and maybe one that prevents movement (or locks the enzyme). all you have to do is have a residue (or two) that physically block one of the pores in the polymerase shape.... | 13:16 |
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| kanzure | a nice way to do this would be to draw the structure for "blocked" and "unblocked" and then write a protein shape solver program that determines which residues are required that can cause both shapes. but this is only for nucleotide incorporation; this does not cover the required slowness or ratcheting single step behavior... | 13:18 |
| kanzure | (if this thing is ultimately super slow, that's probably OK too, because you can multiplex a few million of these on a chip) | 13:18 |
| nmz787 | kanzure: basically take what cambrian was doing (on broad scale, nothing about laser related buzzwords) but simplify and make micro/nano scale... then you can swap around parts like the synthesizer to try different chemistries or enzyme based methods | 13:26 |
| nmz787 | as long as you have everything else in place on chip (sorting, filtering, discrimination, etc) then you have a self-contained feedback loop and life gets a lot easier | 13:27 |
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| nmz787 | no more running around like a space-chicken with its head cut off | 13:27 |
| * nmz787 needs office ergo tips for home... I think this could be a reason for poor productivity | 13:28 | |
| kanzure | huh? their laser method was useful. certainly faster than robot arms everywhere for pipetting... | 13:29 |
| nmz787 | yeah but it still was stupid overall, it was shoved in too late in the game | 13:31 |
| nmz787 | (the game of overall feedback loop process) | 13:31 |
| kanzure | i thought it was their first thing at all? | 13:31 |
| nmz787 | yeah, but they "built on the shoulders of giants" | 13:32 |
| nmz787 | their overall concept was pretty good, but they didn't invest their tech at the right stage, they added it post-filtration/discrimination | 13:32 |
| nmz787 | even though it was essentially a filtration/discrimination step itself | 13:32 |
| nmz787 | and also robots and pipettes, transfer losses, temporal time requirements, etc | 13:33 |
| * nmz787 shudders | 13:33 | |
| kanzure | you will have to elaborate. t12 is often on irc, he worked for them i think. | 13:33 |
| nmz787 | (figuratively, not literally)( | 13:33 |
| kanzure | i noticed that their thermocyclers were manually operated for some reason. they had a bank of like 12 of them. | 13:33 |
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| kanzure | yashgaroth: thakns for the proposal. will be reading shortly. | 14:51 |
| yashgaroth | righto | 14:52 |
| kanzure | grr google scholar keeps asking me for captchas, then redirects me to a page that says 404 (the one where it's supposed to be captcha answer submission) | 14:53 |
| kanzure | "Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes" http://diyhpl.us/~bryan/papers2/polymerase/Evolution%20of%20tRNA%20nucleotidyltransferases:%20A%20small%20deletion%20generated%20CC-adding%20enzymes.pdf | 15:02 |
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| kanzure | relevant: | 15:05 |
| kanzure | http://diyhpl.us/~bryan/papers2/polymerase/Light-dependent%20RNA%20polymerase%20-%20proposal%20-%20Tom%20Hargreaves.pdf | 15:05 |
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| kanzure | http://diyhpl.us/~bryan/papers2/polymerase/Light%20controlled%20synthesis%20of%20nucleic%20acids%20-%20Pinheiro%20-%202010.pdf | 15:06 |
| kanzure | http://diyhpl.us/~bryan/papers2/polymerase/Remote%20electronic%20control%20of%20DNA%20hybridization%20through%20inductive%20coupling%20to%20an%20attached%20metal%20nanocrystal%20antenna%20-%20Jacobson.pdf | 15:06 |
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| nmz787 | kanzure: have you used this? https://chrome.google.com/webstore/detail/the-great-suspender/klbibkeccnjlkjkiokjodocebajanakg?hl=en | 15:12 |
| nmz787 | .title | 15:12 |
| yoleaux | The Great Suspender - Chrome Web Store | 15:12 |
| kanzure | this was from 6 years ago, http://diyhpl.us/~bryan/papers2/polymerase/Exploring%20template-independent%20polymerases%20for%20automated%20DNA%20synthesis%20-%202010.pdf | 15:13 |
| kanzure | nmz787: nope but looks useful. | 15:14 |
| kanzure | not sure why it suspends with a giant "This tab is suspended" grapihc. why not just use a screenshot of the page at low resolution? | 15:14 |
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| nmz787 | good comment | 15:18 |
| kanzure | shrug, otherwise looks okay to e. | 15:19 |
| kanzure | *me | 15:19 |
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| kanzure | cathal made a bunch of helpful comments about that RNA polymerase proposal, https://groups.google.com/forum/#!topic/enzymaticsynthesis/6GZT8zFNOfo | 15:21 |
| kanzure | yea i guess i haven't paid enough attention to CCA-adding enzyme. | 15:23 |
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| CaptHindsight | If you have 2 uncapped and isolated bases ready to be joined <1nm apart and 1 polymerase molecule confined to a space 1nm^3, what will the coupling reaction time be at ~30C? | 15:34 |
| kanzure | poymerase does a coupling in microseconds | 15:35 |
| kanzure | how do you have an isolated base? | 15:35 |
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| FourFire_ | Greetings all | 15:35 |
| FourFire_ | kanzure: how useful is your recording of all conversations of text? | 15:35 |
| kanzure | pretty useful, i wouldn't remember who any of you assholes are otherwise | 15:36 |
| FourFire_ | how long do you spend per day doing the recording on average? | 15:39 |
| nmz787 | CaptHindsight: polymerase is wayy bigger than 1nm on edge | 15:39 |
| kanzure | FourFire_: probably 20 minutes | 15:39 |
| nmz787 | i think at least 150nm | 15:39 |
| nmz787 | i'd | 15:39 |
| FourFire_ | nmz787: check it in a viewer? | 15:41 |
| nmz787 | yea | 15:42 |
| nmz787 | pdb | 15:42 |
| CaptHindsight | nmz787: the active area of the polymerase is ~1nm from the 2 bases | 15:43 |
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| CaptHindsight | so you just want to measure the reaction time | 15:43 |
| nmz787 | ah, ok, I was misled | 15:45 |
| nmz787 | legend | 15:45 |
| * nmz787 closes PDB, noting they didn't have a scale legend | 15:45 | |
| kanzure | i wonder if you could make an ion beam out of nucleotides. | 15:46 |
| * nmz787 ponders | 15:48 | |
| nmz787 | well a mass-spec is essentially that | 15:49 |
| nmz787 | so I would start to look at those kind of papers | 15:49 |
| nmz787 | the e-field should ensure alignment of the polar axes of each nucleotide in said stream | 15:50 |
| nmz787 | but I don't know if the dipoles of each of the 4 are similar enough | 15:50 |
| nmz787 | and also arranged axially of how the polymer would be formed | 15:51 |
| nmz787 | (thinking if you could 3d print DNA polymer with a multi-ion beam) | 15:51 |
| nmz787 | that would be bitchin just for nerd cred | 15:52 |
| CaptHindsight | http://pasteboard.co/L1DVKB3O.jpg just the area in the red circle is where the action happens (not to scale) | 15:53 |
| nmz787 | hmm, I am seeing some 'solvent dependent' dipole search results... I wonder how you can analyze the dipole then in an e-field... seems like you'd need to induce a tumble in the molecule or something, magnets maybe? | 15:54 |
| nmz787 | CaptHindsight: kanzure already answered your 'how long' question, if that's what you mean | 15:54 |
| CaptHindsight | kanzure: you build an isolation track | 15:56 |
| nmz787 | isolation track meaning? | 15:56 |
| CaptHindsight | track, chamber. tube, tunnel | 15:57 |
| CaptHindsight | like in the pic | 15:57 |
| nmz787 | well at that point, you don't need to control polymerase then... just use tdt and flow in individual nucleotides of your desire | 15:58 |
| nmz787 | .wik tdt | 15:58 |
| yoleaux | "Disambiguation: TDT" — https://en.wikipedia.org/wiki/TDT | 15:58 |
| kanzure | not sure anyone has built channels that tiny. | 15:58 |
| nmz787 | .wik Terminal deoxynucleotidyl transferase | 15:58 |
| yoleaux | "Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells." — https://en.wikipedia.org/wiki/Terminal_deoxynucleotidyl_transferase | 15:58 |
| CaptHindsight | who's following anyone? | 15:58 |
| nmz787 | kanzure: sure people have | 15:58 |
| kanzure | CaptHindsight: well usually you have to pick only 1 impossible thing at a time, rather than multiple impossible things at a time :) | 15:59 |
| nmz787 | kanzure: and we have videos of electrophoresing DNA through them using (YOLO1?) dye | 15:59 |
| kanzure | the impossibilities multiply together and team up against you | 15:59 |
| CaptHindsight | kanzure: your way takes too log | 15:59 |
| nmz787 | I learned of this at least 4 years ago: https://groups.google.com/d/msg/diybio/VkIvXhZJZh8/pXxYUyyroCsJ | 16:00 |
| kanzure | what is the mehtod of isolating a single nucleotide, again? | 16:01 |
| nmz787 | .title http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000398/ | 16:01 |
| yoleaux | DNA translocation through short nanofluidic channels under asymmetric pulsed electric field | 16:01 |
| nmz787 | includes video | 16:01 |
| nmz787 | these were 400nm wide | 16:01 |
| nmz787 | which is wider than the breaking radius (or is it diameter) of DNA which is something like 50nm | 16:02 |
| nmz787 | (the amount a polymer can curve before it snaps) | 16:02 |
| nmz787 | which I feel I've also seen with a video | 16:03 |
| nmz787 | yeah, since that link I just pasted is from 2014 | 16:03 |
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| nmz787 | .title http://pubs.rsc.org/en/Content/ArticleLanding/2010/CS/b820266b#!divAbstract | 16:05 |
| yoleaux | DNA manipulation, sorting, and mapping in nanofluidic systems - Chemical Society Reviews (RSC Publishing) | 16:05 |
| nmz787 | "In this critical review, recent experiments utilizing fluidic systems comprised of nanochannels, nanoslits, nanopores, and zero-mode waveguides for DNA analysis are reviewed (161 references)." | 16:05 |
| nmz787 | bah, this isn't the one, but hey tons of references | 16:06 |
| kanzure | nanonostrils? what? | 16:07 |
| kanzure | oh, nanoslits | 16:07 |
| nmz787 | http://pubs.acs.org/doi/abs/10.1021/ac303074f?src=recsys&journalCode=ancham | 16:08 |
| nmz787 | .title | 16:08 |
| nmz787 | "Nanochannels were fabricated having critical dimensions (width and depth) corresponding to 0.5×, 1×, and 2× the DNA persistence length, or 25 nm, 50 nm, and 100 nm, respectively." | 16:08 |
| kanzure | "Electrokinetically-Driven Transport of DNA through Focused Ion Beam Milled Nanofluidic Channels" | 16:08 |
| kanzure | hm. | 16:08 |
| nmz787 | "The nonintermittent DNA transport through the FIB milled nanochannels demonstrates that they are well suited for use in nanofluidic devices. " | 16:08 |
| kanzure | hmm. | 16:09 |
| CaptHindsight | so only failures with a electrostatic gun to print oligos? | 16:09 |
| nmz787 | damn, this is JM Ramsey, I talked with him in 2011 I think about an internship under him | 16:09 |
| nmz787 | :/ | 16:09 |
| nmz787 | didn't take it because he had no $ | 16:09 |
| CaptHindsight | print/assemble | 16:09 |
| nmz787 | CaptHindsight: what failures? | 16:09 |
| CaptHindsight | no ones tried? | 16:10 |
| kanzure | please restate question in the form of an answer | 16:10 |
| nmz787 | CaptHindsight: people have tried flinging nucleotides into reaction wells | 16:10 |
| nmz787 | wells/vessels | 16:10 |
| nmz787 | or droplets/spots even | 16:10 |
| nmz787 | but that is not what I was thinking re: nucleotide ion beam | 16:11 |
| nmz787 | I am thinking, if you could launce in-vacuuo a nucleotide, and alter it's 3D orientation in-flight, such that the ends of the polymer chain were lined up, + to - | 16:11 |
| nmz787 | then maybe you could just grow a polymer by aiming really well | 16:12 |
| kanzure | kid, there's no way you're that good of a shot | 16:12 |
| nmz787 | haha | 16:12 |
| kanzure | you'll never make it | 16:12 |
| kanzure | it's suicide | 16:12 |
| nmz787 | and single ions probably would be tough | 16:12 |
| nmz787 | but like I said, nerd cred | 16:12 |
| CaptHindsight | why launch them? | 16:13 |
| nmz787 | engineer cred is a whole 'nother level | 16:13 |
| nmz787 | CaptHindsight: that is how ion beams work? | 16:13 |
| kanzure | nmz787: if you have any particularly crazy polymerase ideas, i'm in the right place to propose them and get them monies i think. | 16:13 |
| nmz787 | I was just coming up with ideas re: kanzure's prompt "i wonder if you could make an ion beam out of nucleotides. | 16:14 |
| CaptHindsight | heh, like the year old inkjet quote? | 16:14 |
| nmz787 | " | 16:14 |
| kanzure | yes re: focused ion beam stuff | 16:14 |
| kanzure | ("shoot the nucleotides at the polymerase" and such) | 16:14 |
| kanzure | (other than fluid flow) | 16:14 |
| nmz787 | kanzure: I'd rather PM a real-deal kind of thing... just so it isn't ALL in a single paragraph on this log | 16:15 |
| kanzure | i haven't figured out if anyone hsa done low-molecule water count encapsulation of a single molecule, like femtoliter or yoctoliter or whatever it takes. | 16:15 |
| kanzure | *has | 16:15 |
| nmz787 | yocto would be 1nm cube | 16:16 |
| nmz787 | which is pretty darn small | 16:16 |
| kanzure | a 1 nm cube would have a lot of nucleotides, though | 16:16 |
| kanzure | you could try to do dilution, but that's mosty a statistical magic trick more than it is guaranteeing one molecule per bubble | 16:16 |
| nmz787 | that paper you showed was talking 500 attoliter droplets, at 1 micron diameter... 1 micron cube is 1000 attoliter | 16:16 |
| nmz787 | nah just detect the nucleotide, this has also been done (in terms of sequencing) | 16:17 |
| CaptHindsight | where do the funds come from? | 16:17 |
| kanzure | fluorescence signal detection things? | 16:17 |
| nmz787 | but if you control the valve, no need to discriminate the nucleotide flavr | 16:17 |
| CaptHindsight | and what is wanted in exchange for funds? | 16:17 |
| kanzure | CaptHindsight: me, but the guy i'm hanging out with is raising $250M from a certain chinese institution to fund the human genome project reboot | 16:17 |
| kanzure | well the guy really really wants awesome long-length dna synthesis | 16:18 |
| nmz787 | kanzure: I wonder if the US Govt could stop such a thing... i mean pouring to china... ITAR??? | 16:18 |
| kanzure | ITAR does not cover enzymes :D | 16:18 |
| CaptHindsight | I already get funds from China, well in China for the projects in China, how is this different? | 16:18 |
| FourFire_ | HGP reboot? | 16:19 |
| nmz787 | kanzure: I have contacted ITAR folks before, seems like a grey area | 16:19 |
| CaptHindsight | can you work outside china? | 16:19 |
| kanzure | FourFire_: yes | 16:19 |
| nmz787 | kanzure: bioweapon synthesis | 16:19 |
| kanzure | CaptHindsight: they are OK with funding it, but they want the americans to do the labor | 16:19 |
| kanzure | nmz787: ITAR covers bioweapons? that's such a bummer. | 16:19 |
| CaptHindsight | kanzure: how do they keep control over who they fund? | 16:19 |
| kanzure | CaptHindsight: it will be an org in the US probably, run by this guy and maybe me if i decide to join (not sure my role yet, still trying to figure out the pieces) | 16:20 |
| CaptHindsight | kanzure: what do they want in exchange for funds? | 16:20 |
| kanzure | an electronically (or otherwise) controlled DNA polymerase that synthesizes superlong fragments of DNA | 16:21 |
| kanzure | or other highly efficient DNA synthesis methods that do not require a separate assemby step | 16:21 |
| nmz787 | kanzure: http://counsel.cornell.edu/ITAR/ITAR-summary.html#_CHEMICAL_AND_BIOLOGICAL_AGENTS%20AND%20 | 16:21 |
| CaptHindsight | who owns the ip? | 16:21 |
| kanzure | open-source but owned probably by uh.. i don't know. probably the org. | 16:21 |
| kanzure | that's negotiable | 16:21 |
| kanzure | nmz787: that's so lame, you're ruining my day dude. | 16:21 |
| nmz787 | hahah | 16:21 |
| nmz787 | yeah man, it stopped me 4 years ago from pursuing that big sequencing company in china for $$ | 16:22 |
| kanzure | my dreams of weaponized crispr all flushed down the toilet drain...... | 16:22 |
| kanzure | wasn't a company really | 16:22 |
| nmz787 | BGI? | 16:22 |
| kanzure | pretty sure they are not a company, lemme chex | 16:22 |
| kanzure | "non-governmental independent research institute" | 16:23 |
| nmz787 | "Wang Jian, Yu Jun, Yang Huanming and Liu Siqi created BGI in November 1999[2] in Beijing, China as a non-governmental independent research institute in order to participate in the Human Genome Project as China's representative.[3][4] After the project was completed, funding dried up. So BGI moved to Hangzhou in exchange for funding from the Hangzhou Municipal Government." | 16:23 |
| nmz787 | so now govt I guess | 16:23 |
| kanzure | ".. institute in order to participate in the Human Genome Project as China's representative.[3][4] After the project was completed, funding dried up. So BGI moved to Hangzhou in exchange for funding from the Hangzhou Municipal Government." | 16:23 |
| CaptHindsight | http://en.sinotech.ch/2013/03/nanjing-321-plan/ | 16:23 |
| kanzure | hrm. | 16:23 |
| CaptHindsight | was funded by that one ^^^ | 16:23 |
| nmz787 | huh, different refs on that sentence in wikipedia | 16:24 |
| kanzure | oh BGI is probably doing some of the cloning and crispr stuff lately right? that is good. | 16:24 |
| nmz787 | oh, nevermind | 16:24 |
| nmz787 | CaptHindsight posted that link, not kanzure | 16:24 |
| CaptHindsight | but they just wanted the how | 16:24 |
| kanzure | anyway, "who owns the ip" is a reasonabe question | 16:24 |
| kanzure | *reasonable | 16:24 |
| kanzure | not many details yet | 16:24 |
| kanzure | i think that they should choose to fund non-enzymatic synthesis things as well, not just the enzyme dream | 16:25 |
| nmz787 | what is the better-than-libgen thing? | 16:25 |
| kanzure | scihub? | 16:25 |
| nmz787 | yeah | 16:25 |
| CaptHindsight | funding, office and factory space in exchange for coming by and taking whatever they wanted from your office or factory | 16:26 |
| kanzure | how long did that last you? | 16:26 |
| CaptHindsight | it's still there | 16:26 |
| nmz787 | how well do you live? | 16:27 |
| CaptHindsight | I never stayed long | 16:27 |
| CaptHindsight | the software devs never did any work | 16:27 |
| CaptHindsight | and whatever secret sauce I'd leave would disappear when i wasn't there | 16:28 |
| CaptHindsight | what they thought was secret anyway | 16:28 |
| kanzure | nmz787: send e that PM soon | 16:28 |
| kanzure | *me | 16:28 |
| kanzure | CaptHindsight: besides, aren't you a fan of permissive open-source licensing things? openlunchbox etc. | 16:30 |
| CaptHindsight | kanzure: what do they consider long oligos? | 16:30 |
| kanzure | 1000 bp and higher | 16:30 |
| kanzure | ideally 1 million bp or 1 billion bp | 16:30 |
| CaptHindsight | kanzure: some open, some closed | 16:30 |
| CaptHindsight | fan of both | 16:31 |
| kanzure | just not when it's the chinese? hehe | 16:31 |
| kanzure | kidding. | 16:31 |
| FourFire_ | damn, the computer regulation things are dumb: Fault tolerance and performance categories my computer falls under both. | 16:31 |
| CaptHindsight | i give them things so that I can buy them cheap | 16:31 |
| CaptHindsight | like the inkjet DNA printer | 16:31 |
| CaptHindsight | I'll give them that so they can sell them cheap | 16:32 |
| kanzure | yeah if they want to fund an inkjet DNA printer then i think you should susce that out of them | 16:32 |
| kanzure | although i owuldn't mind paying. originally i was hesitant about the pipetting stuff. and we still need a chemist or biologist to sit there and tweak the whole damn system to make the chemistry work. | 16:32 |
| kanzure | *wouldn't | 16:32 |
| CaptHindsight | others are asking for them now | 16:34 |
| CaptHindsight | so maybe a year or two of those then gen2 | 16:34 |
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| CaptHindsight | I have very faith that anyone is going to develop a fast synthesizer anytime soon | 16:35 |
| CaptHindsight | very little | 16:36 |
| kanzure | slow is OK | 16:37 |
| CaptHindsight | we need fast | 16:38 |
| CaptHindsight | watched too many die | 16:38 |
| kanzure | slow is OK because parallelism -- just run 10000 of them in parallel | 16:39 |
| kanzure | huge throughput | 16:39 |
| CaptHindsight | 10K in parallel is ok if they are tiny | 16:40 |
| CaptHindsight | need some quick way to print synthetic viruses or hybrids as well for delivery | 16:42 |
| kanzure | this guy has been working on synthetic viruses with funding from autodesk research | 16:42 |
| kanzure | he is soon going to treat his first patient (a dog) (these are oncolytic synthetic viruses) | 16:43 |
| kanzure | he wants to scale up his synthetic virus production facility | 16:43 |
| CaptHindsight | when is the nanopore patent up? | 16:43 |
| kanzure | there are any nanopore methods.... you mean oxford nano's? | 16:43 |
| kanzure | *many nanopore methods | 16:44 |
| fenn | electrowetting could solve the massively parallel pipetting problem | 16:44 |
| CaptHindsight | yeah, but the patent on a nanopore as a sensor when DNA gets dragged through it | 16:44 |
| CaptHindsight | i forget how broad it is | 16:45 |
| kanzure | yea i guess you could do gibson assembly reactions with a bunch of electrowetting.... | 16:45 |
| kanzure | i think wanting to avoid assembly is a good sentiment | 16:46 |
| fenn | i feel that it's very important that the cost per genome is 6 million USD | 16:46 |
| fenn | we have the technology gentlemen | 16:46 |
| kanzure | although i suppose inkjet + electrowetting is much more reasonable and less speculative. sad. | 16:46 |
| CaptHindsight | if you pay for all the junk vs just the good parts | 16:46 |
| CaptHindsight | inkjet is like using a stone tool | 16:47 |
| CaptHindsight | but it works for now | 16:47 |
| kanzure | CaptHindsight: fenn's suggestion is to do inkjet printing of DNA synthesis reagents, then use electrowetting-on-dielectric underneath each droplet to move the droplets together for the dna assembly reactions (like golden gate or gibson assembly or whatever) | 16:47 |
| CaptHindsight | yeah, we discussed it last year | 16:47 |
| kanzure | ouch | 16:47 |
| fenn | indeed | 16:48 |
| CaptHindsight | for most gene therapy you are just repairing a gene or two | 16:48 |
| kanzure | god damn it, wtf, a year | 16:48 |
| kanzure | how utterly miserable. that needs to be fixed.... | 16:48 |
| CaptHindsight | heh kanzure you first asked me about the inkjet 2 years ago | 16:48 |
| kanzure | stop you're killing me | 16:49 |
| CaptHindsight | why I'm not waiting for anyone else anymore | 16:49 |
| CaptHindsight | it's like drug co speeds | 16:50 |
| kanzure | to be fair, as for andrew's electronically-controlled polymerase, he and i were first talking about that ~6 years ago--- which is even worse | 16:50 |
| kanzure | and i'm pretty sure i was thinking about it much earlier (as was he and others) | 16:50 |
| kanzure | so all of this has been a long-time coming | 16:50 |
| fenn | ~15 years ago for me | 16:50 |
| kanzure | in 2008 when i joined ellington's lab, one of the grad students (a friend of asciilifeform) proposed this method - http://diyhpl.us/~bryan/papers2/polymerase/2008-06-06_beta_clamp.png | 16:51 |
| CaptHindsight | it's all your fault | 16:51 |
| kanzure | unfortunately i did not take good notes, and that diagram was all that i remembered | 16:51 |
| kanzure | it's definitely all my fault but that's okay. | 16:51 |
| CaptHindsight | I thought you guys would be done by now | 16:51 |
| kanzure | we have certain pieces but not all of them | 16:52 |
| kanzure | and at the moment less than infinite funding, so have to be careful about spend | 16:52 |
| CaptHindsight | I figure once repet is up it should fund itself :) | 16:53 |
| kanzure | i think korea already has one of those | 16:53 |
| kanzure | sooam | 16:53 |
| kanzure | http://en.sooam.com/dogcn/sub01.html | 16:54 |
| CaptHindsight | well they just clone, ppfftt | 16:54 |
| CaptHindsight | we have the added value of modifying | 16:54 |
| fenn | is "repet" the name of a company? | 16:54 |
| kanzure | "When your dog has passed away, <b style="color:#f90;">DO NOT</b> place the cadaver inside the freezer. Then, patiently follow these steps: 1. Wrap the entire body with wet bathing towels. 2. Place it in the fridge(not the freezer) to keep it cool. * Please take into account that you have approximately 5 days to successfully extract and secure live cells. Email: admin@sooam.org Telephone: +82 70 7722 9354" | 16:55 |
| kanzure | fenn: it's from total recall | 16:55 |
| kanzure | or er, sixth day | 16:55 |
| CaptHindsight | https://www.youtube.com/watch?v=CtoLvF_TlSA | 16:55 |
| CaptHindsight | but no mind swap | 16:55 |
| kanzure | .title https://www.youtube.com/watch?v=CtoLvF_TlSA | 16:56 |
| yoleaux | The 6th Day - RePet Infomercial - YouTube | 16:56 |
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| kanzure | i was thinking of "rekall" https://www.youtube.com/watch?v=lak6Nf-aSvQ | 16:59 |
| fenn | wow that was a very long commercial | 16:59 |
| kanzure | movies are mostly commercials anyway, so it fits | 17:00 |
| CaptHindsight | the remake is already a few years old | 17:01 |
| CaptHindsight | Total Recall and the 6th Day were 10 years apart | 17:02 |
| CaptHindsight | talk about time flying | 17:02 |
| kanzure | in this channel, we mostly experience time warps and time bubbles, not so much time flight | 17:02 |
| kanzure | time traps, and such. | 17:03 |
| CaptHindsight | we were growing hair back on cadaver scalps in the mid 80's | 17:03 |
| kanzure | is that some sort of sick necro thing? | 17:03 |
| CaptHindsight | flipping hair back on | 17:03 |
| CaptHindsight | growing teeth as well | 17:03 |
| CaptHindsight | I thought by 2000 we'd have all this worked out | 17:04 |
| kanzure | CaptHindsight: dunno if you saw yesterday but this guy wants to do disposable one-off "epi pens" that do dna synthesis followed by synthetic virus manufacturing and finally electroporation or introduction of the virus into a body, as a gene therapy programing pencil tool | 17:04 |
| kanzure | *programming | 17:04 |
| kanzure | fenn: surely there is a reason why church has not already done inkjet + electrowetting? | 17:05 |
| CaptHindsight | pen, tri-corder, magic slipper | 17:05 |
| kanzure | no the tricorder stuff was bull from day one, including the xprize | 17:06 |
| CaptHindsight | I wonder who will get it working first? | 17:06 |
| CaptHindsight | just a name for a gadget | 17:06 |
| nmz787 | kanzure: sent, plz try to at least keep me in the loop if they like it, ideally this would be a keystone in my career path, as a core component of many zany synbio ideas... so I'd really like to be involved intimately | 17:06 |
| kanzure | CaptHindsight: "scanadu" was the company name | 17:07 |
| kanzure | nmz787: haha don't worry about your career, you are in good hands without this | 17:07 |
| kanzure | *even without this | 17:07 |
| CaptHindsight | Scamadu | 17:07 |
| nmz787 | CaptHindsight: my thougts exactly | 17:07 |
| CaptHindsight | like poopstarter | 17:08 |
| kanzure | i remember hearing so much drama about scanadu. happy that i currently remember none of the details. yay. | 17:08 |
| * nmz787 goes to drink tea on the porch | 17:08 | |
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| kanzure | nmz787: my concern is how do you get a stream of single confined nucleotides, precisely? | 17:11 |
| kanzure | nmz787: you want fluorescence detection of nucleotides to decide whether an injector is loaded correctly with a single nucleotide ? | 17:12 |
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| kanzure | i wouldn't be concerned about enzyme size, btw. this can be fixed. enzymes can be given lots of additional volume. | 17:13 |
| nmz787 | kanzure: as it says, the amount isn't dependent on single nt input | 17:15 |
| nmz787 | kanzure: flourescense works with dyes, which adds to reagent cost and general procurement requirements... could probably mess with downstream stuff maybe too | 17:17 |
| nmz787 | raman is basically looking at self-fluorescense, but requires optics | 17:17 |
| nmz787 | electronic methods would be first method to attempt using | 17:17 |
| kanzure | "the amount isn't dependent on single nt input" | 17:17 |
| * kanzure looks again | 17:17 | |
| nmz787 | as they are cheapest and super easy to implement (two wires) | 17:17 |
| nmz787 | "from a single molecule up to an amount tolerable by | 17:18 |
| nmz787 | downstream discriminators" | 17:18 |
| kanzure | oh, you mean, just redo everything until it gets it right? | 17:18 |
| kanzure | this will only work for short fragments :) | 17:18 |
| nmz787 | for a single oligo, yeah, if error->redo | 17:18 |
| kanzure | for 30 bp it's already 30^4 plus it's randomized so it's a random walk | 17:19 |
| kanzure | no i think it's 30^4 factorial | 17:19 |
| kanzure | .wa (30^4)! | 17:20 |
| nmz787 | hmm? | 17:20 |
| kanzure | the number of attempts, with random incorporation | 17:20 |
| kanzure | oh sorry, i forgot that you can select which nucleotide type | 17:20 |
| kanzure | so the error is mostly "repeats" | 17:20 |
| nmz787 | well you could also foreseee optimizations such as a deletion might be able to be tried again for addition | 17:20 |
| nmz787 | or an over-addition might get a snip treatment from a cutter-ase | 17:21 |
| kanzure | the image diagram i pasted above has a method of pausing tdt | 17:21 |
| nmz787 | whatever they're called | 17:21 |
| nmz787 | endonuclease | 17:21 |
| kanzure | http://diyhpl.us/~bryan/papers2/polymerase/2008-06-06_beta_clamp.png | 17:21 |
| nmz787 | you could probably pause it simply by cooling and heating | 17:21 |
| kanzure | oh sorry, no nevermind. | 17:21 |
| * nmz787 looks for link | 17:21 | |
| nmz787 | got it | 17:22 |
| kanzure | actually it would be better if it's a one-shot enzyme, e.g. the enzyme degrades after its only use | 17:22 |
| nmz787 | why? that sounds terrible | 17:22 |
| kanzure | because if it doesn't work after incorporation, you just add another one, right? | 17:22 |
| nmz787 | you just threw this idea out the economically feasible window, i thin | 17:22 |
| nmz787 | k | 17:22 |
| kanzure | repeats are bad though | 17:23 |
| nmz787 | but you have moved the DNA away from the enzyme at that point | 17:23 |
| kanzure | yes true | 17:23 |
| kanzure | i don't think we have any enzymes that can cleave a variable number of repeated nucleotides | 17:23 |
| nmz787 | so you can just use a different enzyme to remove the double (my optimization thought, not required for first-pass waste-a-ton-but-still-save-cause-micro-nano-scale) | 17:23 |
| kanzure | *any exonucleases | 17:24 |
| nmz787 | yeah so its an optimization thought, not requirement I think | 17:24 |
| kanzure | the way to prevent repeats is to have a source of single nucleotides | 17:24 |
| kanzure | single nucleotides should be a requirement here | 17:24 |
| nmz787 | plus I'm sure you could use any old exonuclease and just laser pulse it to heat-start it | 17:24 |
| kanzure | you could modify tRNA synthetase or some other enzyme to hold a single nucleotide, which makes it easier to physically manipulate and separate into bubbles | 17:25 |
| nmz787 | kanzure: well not necessarily, it can be done in parallel... it's a game of optimization, but I agree single nt input would be optimal if it wasn't terribly troublesome/expensive | 17:25 |
| nmz787 | bubbles for what? | 17:25 |
| kanzure | well you would have a bubble (water-oil immersion, like w-o-w or o-w-o or something) to separate a single nucleotide from the next one | 17:26 |
| nmz787 | oh, I was not thinking that | 17:26 |
| kanzure | yes well the difficulty is how to produce bubbles where you know a single nucleotide is inside | 17:26 |
| nmz787 | yeah, just skip the bubbles then, makes the problem half as hard (?) | 17:27 |
| kanzure | with enzymes inside the bubbles you can know that one enzyme has one nucleotide attached | 17:27 |
| kanzure | and you can attach an enzyme to a giant bead or giant nanoparticle, things like that | 17:27 |
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| nmz787 | too many dynamic variables for me to "know" intuitively | 17:27 |
| nmz787 | vs hard-constraints of spatial constructs | 17:28 |
| kanzure | nucleotides are really really small molecules | 17:28 |
| nmz787 | we can fab stuff small enough, there is no need to make things larger | 17:28 |
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| nmz787 | all you need to know is "is there a 10-mer present" etc... the rest is recursion | 17:29 |
| nmz787 | with an occasional 'yield' | 17:29 |
| kanzure | yes but with repeats, the number of retries explodes and makes the whole thing infeasible for long oligos | 17:29 |
| nmz787 | I still bet this is way cheaper by several orders of magnitude than what is available today | 17:30 |
| nmz787 | I think I have calculated this with errors included before | 17:30 |
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| kanzure | you could argue to me: we'll find/make an enzyme that only incorporates once per minute. i would accept that. we could make an enzyme like this. | 17:30 |
| nmz787 | I mean, just use error rates for shitty-today synthesis and scale the reagent cost alone | 17:30 |
| kanzure | one minute is enough time to wash away the other nucleotides | 17:30 |
| nmz787 | incorporation is controlled by heat, so there is your control (I think you showed me papers of pulsing with NIR laser at an enzyme to control temp and thus reaction rate?) | 17:31 |
| kanzure | is it a nanopore? or just free floating in your model? | 17:31 |
| nmz787 | it meaning? | 17:31 |
| kanzure | the enzye | 17:31 |
| kanzure | enzyme | 17:32 |
| nmz787 | it just floats and is kept in a strainer | 17:32 |
| nmz787 | which has a bunch of pores small enough so it doesn't escape through | 17:32 |
| kanzure | heat is not good because it could have already incorporated another nucleotide by the time you have flashed a laser at it | 17:32 |
| nmz787 | easily fabbed, or purchased for cheap if a sandwich device could be made | 17:32 |
| kanzure | you need it to just, by default, not incorporate another nucleotide somehow. | 17:32 |
| nmz787 | yeah, keep cold, pulse laser? | 17:33 |
| kanzure | yes i understand the strainer concept. the enzyme can be made larger if necessary, i don't think that's a proble. | 17:33 |
| nmz787 | if the refrigerant had a high enough heat capacity, should be able to pull temp down quickly after turning off laser | 17:33 |
| kanzure | pulse the laser but within the 2 microseconds it took you to decide that, a repeat could have been added... | 17:33 |
| nmz787 | the key thing I guess would be diffusion speed vs pulldown of temp speed | 17:33 |
| nmz787 | the enzyme DOESNT need to be bigger, lol, you're making things harder for no apparent reason | 17:34 |
| nmz787 | (and also the distribution of nt in solution, i.e. the concentration) | 17:34 |
| kanzure | well i don't consider that detail to be hard, but yes i agree with you that a strainer works | 17:34 |
| nmz787 | .title temwindows.com | 17:34 |
| kanzure | concentration is not a guarantee really | 17:34 |
| nmz787 | has them for like $30 | 17:34 |
| nmz787 | yeah but the point is it shouldn't matter that much, since you're watching for the right size molecules. | 17:35 |
| xentrac | .title http://temwindows.com/ | 17:35 |
| nmz787 | if you command 10 additions, then discard any that don't end up 10 when you check | 17:35 |
| * xentrac whacks yoleaux with a stick and then notices it's not there | 17:35 | |
| kanzure | ok good point, size is the only thing you care about. but nuclease is still problematic. and starting over when you are at nucleotide number 300,210 is a real bummer. | 17:35 |
| nmz787 | it will not be WORSE than today's solid-phase phosphoramidite efficiencies | 17:36 |
| kanzure | s/size/length | 17:36 |
| nmz787 | nah, you yield small fragments into a pool for gibson assembly or something (maybe serial gibson in a nanochannel for reducing 3D looping of DNA onto itself, making assembly difficult) | 17:37 |
| nmz787 | (a pool, or a stream of oil water oil drops) | 17:37 |
| nmz787 | I don't like the idea of adding more chemicals (oil) if I can think of another way | 17:37 |
| * nmz787 attempting to KISS even though super-not-S | 17:38 | |
| kanzure | i think it's a little concerning that search queries like "single molecule dilutions" don't turn up much | 17:40 |
| nmz787 | I think it's too generic a search | 17:40 |
| nmz787 | of course you can do it, it is written into the equations | 17:41 |
| nmz787 | probably try specifying more application keywords | 17:41 |
| nmz787 | dilution isn't hard for single-molecules, it is detection | 17:41 |
| nmz787 | (why I think work would be ideal for this kind of device, because on-chip preamps and such are probably prudent) | 17:42 |
| nmz787 | work meaning my employer | 17:42 |
| kanzure | yes there's a semiconductor consortium involved in some synthetic biology things (i am getting more info) | 17:43 |
| nmz787 | https://www.jstage.jst.go.jp/article/bunsekikagaku/64/6/64_431/_pdf | 17:43 |
| nmz787 | hah, in japanese | 17:43 |
| kanzure | (semisynbio) | 17:43 |
| nmz787 | yeah | 17:43 |
| nmz787 | I think I posted about them a month or so ago | 17:43 |
| nmz787 | strange the abstract is in english https://www.jstage.jst.go.jp/article/bunsekikagaku/64/6/64_431/_article | 17:43 |
| kanzure | abstract is at the bottom of your pdf link | 17:44 |
| nmz787 | yeah, here http://gnusha.org/logs/2016-06-01.log | 17:45 |
| kanzure | heard about it through your employer? | 17:46 |
| nmz787 | yeah | 17:48 |
| kanzure | i guess you could use crispr to fix repeats | 17:49 |
| kanzure | if concentration solves the whole problem then you're done, i think. | 17:50 |
| Proteus1 | alife 2016 - 15th conference on the synthesis and simulation of living systems: https://mitpress.mit.edu/sites/default/files/titles/free_download/9780262339360_ALIFE_2016.pdf | 17:59 |
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| nmz787 | "Katie Bentley: Do Endothelial Cells Dream of Eclectic Shape?" | 18:04 |
| nmz787 | wut | 18:04 |
| nmz787 | "Seth Bullock: ALife as a Model Discipline for Policy-Relevant Simulation Modelling: Might “Worse” Simulations Fuel a Better Science-Policy Interface? " | 18:05 |
| nmz787 | hmm, scare tactics essentially? | 18:05 |
| nmz787 | oh, kanzure, in the settings of that extension it has a "Enable screen capturing" checkbox | 18:11 |
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| nmz787 | "There are additional efforts being made to map or sequence DNA molecules in nanochannels using electronic rather than fluorescent means. Liang and Chou144 have used NIL and shadow evaporation techniques to fabricate 50 mm long fluidic channels 45 nm in width and depth with a pair of nanowire electrodes transverse to the channel resulting in a metallic gap down to 9 nm in width and 16 nm in height. This allows the measurement of ionic ... | 18:46 |
| nmz787 | ... conductance perpendicular to the DNA backbone as the DNA is electrophoresed through the gap. With 1.1 kbp DNA molecules flowing through the channel, reductions in the transverse ionic current of B350 pA were observed for typical duration times of B100 mS attributed to blockage of the gap by the insulating DNA molecule. Somewhat large variation in this duration time needs to be further understood and the gap reduced before one can expect ... | 18:46 |
| nmz787 | ... sequence specific information to be obtainable." | 18:46 |
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