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| abetusk | http://www.gaudi.ch/GaudiLabs/?page_id=392 | 01:03 |
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| abetusk | saran wrap + rain x | 01:05 |
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| streety | kanzure, I think it is one bead. There will be some droplets with zero or more than one, but it is the droplets with just one bead that are the useful droplets | 05:02 |
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| fenn | .wik ibutamoren | 05:47 |
| yoleaux | "Ibutamoren (INN) (developmental code names MK-677, MK-0677, L-163,191) is a non-peptidic, potent, long-acting, orally-active, and selective agonist of the ghrelin/growth hormone secretagogue receptor (GHSR) and a growth hormone secretagogue, mimicking the growth hormone (GH)-stimulating action of the endogenous hormone ghrelin." — https://en.wikipedia.org/wiki/Ibutamoren | 05:47 |
| fenn | "Restoring HGH in "normally-aging" people is not a function that the Food and Drug Administration (FDA) considers to be a legitimate function of a medicine; therefore, Merck (the pharmaceutical company) stopped all further development of MK-0677." | 05:49 |
| EnLilaSko | Ibutamoren seems to promote a more bleed-like GH release, meh | 05:50 |
| fenn | sounds like sour grapes | 05:51 |
| kanzure | hm | 05:54 |
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| kanzure | http://untappedcities.com/2013/03/15/nycs-pneumatic-tube-mail-network/ | 06:08 |
| kanzure | .title http://www.gaudi.ch/GaudiLabs/?page_id=392 | 06:12 |
| yoleaux | Welcome to » OpenDrop – Digital Microfludics Plattform | 06:12 |
| kanzure | i'm still not convinced about electrowetting as a viable method for our projects | 06:12 |
| fenn | i think there would be too much cross contamination | 06:13 |
| kanzure | do you think cross-contamination matters for the ligase steps? | 06:14 |
| kanzure | also, you could just leave open-spaces i think | 06:14 |
| fenn | the third pneumatic cylinder "had a black cat in it, for reasons unknown" | 06:15 |
| fenn | early quantum cryptography experiment | 06:15 |
| kanzure | .title https://www.youtube.com/watch?t=71&v=C677yPYXWIs | 06:16 |
| yoleaux | Electrowetting - Digital Microfluidics on Printed Circuit Board - Prototype - YouTube | 06:16 |
| kanzure | .title https://www.youtube.com/watch?v=53JiNYVzzHM | 06:16 |
| yoleaux | OpenDrop Digital Microfluidics on Printed Circuit Board - YouTube | 06:16 |
| kanzure | http://www.bioflux.eu/ | 06:18 |
| kanzure | http://hackteria.org/wiki/Elektrowetting | 06:18 |
| fenn | it looks like the drop is too big | 06:19 |
| kanzure | apparently this is all recent http://hackteria.org/wiki/index.php?title=Elektrowetting&action=history | 06:20 |
| kanzure | and here is their source code and schematics https://github.com/waagsociety/OpenFluxl | 06:20 |
| kanzure | https://github.com/waagsociety/OpenFluxl/blob/master/Components/OpenFluxl%20Components%20v.0.1.md | 06:21 |
| kanzure | oh this is pieter boheeman... i see. | 06:21 |
| kanzure | boheemen. | 06:21 |
| fenn | so the idea is that you print your oligos directly on one of these arrays? | 06:22 |
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| kanzure | yes | 06:23 |
| kanzure | but i still like the idea of just pushing the droplets around with a giant metal stick | 06:24 |
| fenn | yeah that's super scalable~~~ | 06:24 |
| kanzure | web scale | 06:24 |
| fenn | wouldnt you need like 100,000 sticks all perfectly aligned | 06:25 |
| kanzure | you could also have a giant metal comb and push the droplets around like you're cutting lines of cocaine | 06:25 |
| fenn | ok so that's only 1000 razor blades at least | 06:26 |
| fenn | .c 100000^0.5 | 06:26 |
| kanzure | hm | 06:26 |
| yoleaux | 100000^(1/2) = 100 sqrt(10) ≈ 316.227766016837933 | 06:26 |
| fenn | so the big idea behind cambrian genomics was to select known good sequences early in the assembly process by doing on-slide sequencing; droplet manipulation could work just as well as bead manipulation | 06:28 |
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| fenn | instead of firing beads around you just move droplets around | 06:29 |
| fenn | unfortunately then your oligo synthesis substrate becomes a giant microfluidic chip | 06:29 |
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| fenn | lately i seem to be having trouble stating things in a straightforward way | 06:30 |
| kanzure | electrowetting at least does not have all the other problems of microfluidics | 06:30 |
| fenn | it's definitely easier to think about | 06:31 |
| kanzure | i am ok with lasers but where are you going to be launching stuff, and then after it lands how will you combine some of the results? | 06:31 |
| fenn | can EWOD arrays handle droplets of varying size? | 06:31 |
| fenn | i remember one demo where they separated droplets from a "reservoir" that was made up of many pixels | 06:32 |
| kanzure | .ttile https://www.youtube.com/watch?v=jpbUvSyeRg0 | 06:33 |
| kanzure | yoleaux: i command you! | 06:34 |
| fenn | i dont get why each EWOD pixel requires its own dedicated transistor | 06:34 |
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| kanzure | .title https://www.youtube.com/watch?v=jpbUvSyeRg0 | 06:34 |
| yoleaux | Droplet Dispensing and Mixing in Digital Microfluidics - YouTube | 06:34 |
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| fenn | that's the wrong video | 06:35 |
| fenn | .title http://youtu.be/WxpQyqoukpc | 06:36 |
| yoleaux | Programmable large area digital microfluidic array with integrated droplet sensing for bioassays - YouTube | 06:36 |
| fenn | ok i guess they are moving the large drops around too | 06:36 |
| kanzure | ah i remember their test setup (exposed electrodes) https://www.youtube.com/watch?v=k9YE4jf-wzo | 06:40 |
| fenn | "FluxMux-Device | 06:40 |
| fenn | Based on two crosswise flat ribbon cable the FluxMux device is an easy way to create a digital microfluidic device. An array of power leds shines through the grid to make drops visible. | 06:40 |
| fenn | The control is multiplexed. | 06:40 |
| fenn | so i dont get why they think they need individual transistors | 06:40 |
| kanzure | .ttile https://www.youtube.com/watch?v=hVAa41qTIqg | 06:41 |
| kanzure | .title https://www.youtube.com/watch?v=hVAa41qTIqg | 06:41 |
| yoleaux | Two-plate digital microfluidics for dispensing, mixing, and merging droplets - YouTube | 06:41 |
| fenn | instead of printing directly on the array you can print on a glass slide, then flip the slide over and put it on the array | 06:42 |
| fenn | i'm not sure how to get a thin layer of oil on top of tiny droplets without disturbing the position of the droplets | 06:43 |
| fenn | is the oil really necessary? | 06:44 |
| kanzure | well you were complaining about evaporation | 06:45 |
| fenn | yes but that may not be a problem if there's a cover slide | 06:45 |
| kanzure | well it would suck for adding more ligase reaction reagents later | 06:46 |
| kanzure | i guess oyu could have "supply droplets" that you pre-printed | 06:46 |
| kanzure | for later. | 06:46 |
| kanzure | (unless you had a hole in the cover slide) | 06:46 |
| fenn | the oligos would be dried up by the time you remove them from the machine, so you will have to add water and ligase anyway to re-dissolve them. and also probably add something to cleave the oligos from the surface | 06:47 |
| fenn | so there would have to be 2 inkjet printers | 06:47 |
| fenn | one to make the oligos, and one to make reaction droplets | 06:48 |
| kanzure | do you mean two printheads? | 06:48 |
| fenn | no, because one of them must be maintained in a completely water-free environment, and the other one is shooting water | 06:48 |
| fenn | yay chemistry! | 06:48 |
| fenn | you can get away with a lower resolution on the second printer though, because you're going to be combining 10+ droplets anyway | 06:49 |
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| kanzure | if the surface is hydrophobic then i think you're better able to remove the water, as long as there's no evaporation | 06:51 |
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| fenn | maybe you can use an LCD TFT for the array | 06:53 |
| fenn | if the oligos are cleaved and hybridized on the array, you can put down a sparse grid of small droplets, then move the droplet around to each oligo in sequence | 06:59 |
| fenn | this makes better use of the printed area because otherwise you'd have to waste a lot of margin between large circular drops, because it's printed on a square grid and circles don't tile very well | 07:00 |
| fenn | it would be really nice to be able to separate long oligos form short ones directly on the array | 07:02 |
| fenn | otherwise reaction byproducts will build up and make a mess | 07:02 |
| fenn | mis-hybridized strands that got ligated, or badly synthesized oligos with no overhanging ends | 07:02 |
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| fenn | i made a drawing http://fennetic.net/irc/dna_printer_EWOD_oligo_resuspension.png | 07:46 |
| fenn | at each step you raise temperature to 95C then slowly lower it to 60C to allow stringent hybridization, then move to the next pixel; step 10 is to combine the nearest 9 droplets | 07:48 |
| fenn | it's worth noting that each pixel can be much larger than the size of the individual oligo sequence spots, so you could crap 10 of them in each pixel for example | 07:57 |
| fenn | cram* heh | 07:58 |
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| fenn | then in the droplet there would be a population of 1,2,3...9 double strands each 225bp long (25 bp single stranded overhang on either end, assuming 50bp oligos) | 08:01 |
| fenn | you want to combine them in the last step because short strands have lower melting points than long strands | 08:02 |
| fenn | maybe it's easier to move the droplet in an outward spiral, because then you don't have to traverse over oligos on your way to the droplet combination location | 08:10 |
| fenn | actually it doesn't have to be a spiral at all, it can be linear | 08:11 |
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| streety | after you have combined the closest two 9's, would you not then need to repeat with the closest two 10's, 11's etc? | 08:32 |
| streety | would you leave some pixels blank to allow for merging corridors? | 08:33 |
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| fenn | hierarchical linear combination http://fennetic.net/irc/dna_printer_EWOD_oligo_resuspension_linear.png | 08:39 |
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| streety | speedy diagramming | 08:40 |
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| fenn | i had been drawing it already | 08:42 |
| kanzure | oh look a drawing | 08:43 |
| kanzure | that's not fair why do you have a good drawing | 08:43 |
| fenn | because i used inkscape instead of gimp | 08:43 |
| kanzure | fucker | 08:44 |
| kanzure | yeah i probably should have realized something was wrong when i was using gimp instead of inkscape... | 08:44 |
| kanzure | and you are not worried about water evaporation? | 08:44 |
| kanzure | i'm not sure whether ewod works for small droplets on big electrodes | 08:45 |
| fenn | having a slide on top reduces the surface area significantly | 08:45 |
| fenn | i don't know if evaporation will be a problem or not | 08:45 |
| fenn | 95C is pretty hot | 08:46 |
| kanzure | isn't there some trick to this like "as you increase the temperature, lower the pressure to avoid evaporation"? | 08:46 |
| fenn | raise partial pressure of h2o | 08:46 |
| fenn | hmm | 08:47 |
| kanzure | what was the drop transfer mechanism you wanted? | 08:48 |
| kanzure | i don't see how you would transition from the inkjet to this | 08:48 |
| fenn | which inkjet | 08:49 |
| kanzure | "the surface already has electrodes when you synthesize the oligos the first time around, so you just transfer the plate into the other machine and add a coverslip after printing water/reagents"? | 08:49 |
| fenn | yes | 08:49 |
| kanzure | did this require direct contact with the electrode? | 08:50 |
| fenn | they have to be pretty close to the surface but there is a dielectric film over the electrodes(?) | 08:50 |
| kanzure | ok so the film would have to be compatible with the phosphoramidite chemistry, acetonitrile, etc. | 08:51 |
| fenn | yes | 08:51 |
| kanzure | so does this mean we need an electrode grid the size of the inkjetted spot array? | 08:53 |
| fenn | yes, i think an LCD panel could work but it would need new electronics to drive it at high voltage | 08:53 |
| fenn | something like a TI-85 calculator display would be easier to work with because of the larger dot pitch, but those dont typically have TFT built in | 08:55 |
| kanzure | yes we have been over this before i think | 08:55 |
| kanzure | http://teitell-lab.com/wp-content/uploads/2014/02/2010_4_Park.pdf | 08:55 |
| fenn | alternatively we could make a PCB that just does this | 08:55 |
| fenn | the electronics will be simpler because of the linear layout | 08:56 |
| fenn | you only need 3 phases | 08:56 |
| fenn | they can all be driven in parallel at the same time | 08:57 |
| fenn | so i guess that's only 3 transistors | 08:57 |
| fenn | instead of 1 million or whatever | 08:58 |
| kanzure | .title https://www.youtube.com/watch?v=u_Be2awFf0c | 08:59 |
| yoleaux | Single-sided continuous optoelectrowetting (SCOEW) for droplet manipulation with light patterns - YouTube | 08:59 |
| kanzure | (related to that last paper link) | 08:59 |
| kanzure | not a good video | 09:00 |
| fenn | "optical electrowetting" sounds like just laser tweezers | 09:01 |
| kanzure | here's one where they move droplets using suction https://www.youtube.com/watch?v=Qjeb_3y6RU8 | 09:01 |
| kanzure | huh... https://www.youtube.com/user/labonachipVideos/videos | 09:02 |
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| fenn | down the microfluidics rabbit hole... | 09:03 |
| kanzure | "Instantaneous room temperature bonding of a wide range of non-silicon substrates with poly(dimethylsiloxane) (PDMS) elastomer mediated by a mercaptosilane" https://www.youtube.com/watch?v=FIwX28dRkVI | 09:03 |
| kanzure | yes perhaps i shouldn't look | 09:03 |
| kanzure | heh, a "bubble pump" https://www.youtube.com/watch?v=3WOop8-jcjY | 09:03 |
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| kanzure | .title https://www.youtube.com/watch?v=a-28gvw8WkQ | 09:07 |
| yoleaux | Micro Propulsion by Acoustic Bubble for Navigating Microfluidic Spaces - YouTube | 09:07 |
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| fenn | its like a pulse jet | 09:08 |
| fenn | water gets sucked in from all directions but goes out in only one direction | 09:09 |
| fenn | ok so maybe this could be done with a bunch of razor blades | 09:10 |
| fenn | instead of electrowetting | 09:11 |
| fenn | electrowetting actually seems simpler though | 09:11 |
| kanzure | yes it is closer to solid state stuff | 09:11 |
| kanzure | and more solid state stuff is better and makes life simpler | 09:12 |
| kanzure | oh, razor blades are also solid state | 09:12 |
| kanzure | realistically we are not going to have a 2000-drop wide razor blade that we move only 50 microns (so that it doesn't slide them into the next line/row) | 09:13 |
| kanzure | unless we get some really small paperclips | 09:13 |
| kanzure | this wont work for our purposes but there was a way of grating a surface so that droplets move at variable speeds in certain directions. if you could control that sufficiently well you could have it also timed to the ligation protocol steps. | 09:16 |
| kanzure | (they would move up the gradient or grooves because of surface tension caused by the patterning on the surface) | 09:17 |
| juri_ | have you thought of using the razorblade as 'ground', and forcing the droplets around that way? | 09:18 |
| kanzure | the droplets have a diameter of <20 microns | 09:18 |
| juri_ | ok, so the finest razorblade is just too blunt. | 09:20 |
| juri_ | even if you were trying to hold it a fraction from the surface, getting it between droplets just isn't feasable. | 09:20 |
| juri_ | and i can't find wire in sizes that small... | 09:27 |
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| kanzure | man if star wars 7 doesn't have a new superweapon (or the death star) i'm going to be grump | 10:10 |
| chris_99 | heh | 10:16 |
| streety | does anyone have any recommendations for a good laptop? My backup has just failed, and my main is less reliable than I would like | 10:33 |
| chris_99 | i just saw https://news.ycombinator.com/item?id=9911699 on HN, may be helpful? | 10:33 |
| chris_99 | .title | 10:33 |
| yoleaux | Ask HN: Most hacker friendly laptop in market(2015) | Hacker News | 10:33 |
| kanzure | streety: thinkpad w520 has been serving me well. there'a w530 also. | 10:34 |
| streety | very relevant, thanks | 10:35 |
| kanzure | oh they claim "HP Zbook 15 G2" can do 64 GB RAM. hmm. maybe i should upgrade. | 10:36 |
| kanzure | hmm hp seems to disagree | 10:37 |
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| justanotheruser | I have a w530, love everything about it except the battery life | 10:47 |
| kanzure | get battery extension pack thingy | 10:49 |
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| fenn | wow at the end of the Park SCOEW paper they just use a LCD monitor to move droplets around | 11:02 |
| fenn | also i am a dumbass and didn't realize the obvious implication that a droplet handling system can make a bunch of small droplets from one large blob, so you don't need a second inkjet printer | 11:05 |
| fenn | it might be possible to get rid of the first inkjet printer too | 11:16 |
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| kanzure | fenn: with a large enough blob i guess you could make sub droplets in parallel? | 11:48 |
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| kanzure | finger actuation of electrowetting / EWOD by using a piezoelectric element http://pubs.rsc.org/en/content/articlelanding/2014/lc/c3lc51223a | 11:53 |
| kanzure | "Instead of requiring an external power supply, our F-DMF uses piezoelectric elements to convert mechanical energy produced by human fingers to electric voltage pulses for droplet actuation. Voltage outputs of over 40 V are provided by single piezoelectric elements, which is necessary for oil-free EWOD devices with thin (typically <1 μm) dielectric layers. Higher actuation voltages can be provided using multiple piezoelectric elements ... | 11:54 |
| kanzure | ... connected in series when needed. Using this energy conversion scheme, we confirmed basic modes of EWOD droplet operation, such as droplet transport, splitting and merging. Using two piezoelectric elements in series, we also successfully demonstrated applications of F-DMF for glucose detection and immunoassay." | 11:54 |
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| kanzure | .title http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079775/ | 11:57 |
| yoleaux | Picoliter DNA Sequencing Chemistry on an Electrowetting-based Digital Microfluidic Platform | 11:57 |
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| kanzure | .title lindsay salis | 12:01 |
| yoleaux | kanzure: Sorry, that command (.title) crashed. | 12:01 |
| kanzure | fjoiefjeqreq | 12:01 |
| kanzure | .title https://www.youtube.com/watch?v=UQbqvqn7BSE | 12:01 |
| yoleaux | Active thermal management of on-chip hot spots using EWOD-driven droplet microfluidics - YouTube | 12:01 |
| kanzure | is that something i should expect to cool the chip? | 12:01 |
| CaptHindsight | I wonder how many Irish Folk Dancers it would take to power a cluster of these arrays? | 12:01 |
| kanzure | "biomems" lectures https://www.youtube.com/playlist?list=PLqYqvTonHe8-ok_aryb6UQlshRMVL_96L (27 videos) | 12:08 |
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| streety | My current thoughts on a replacement laptop are that the HP Zbook at 6lb is way too heavy. I'm actually tempted to go as light as possible this time around and see how it works for me. The laptop that has just failed was a lenovo but it took a lot of punishment so would definitely consider sticking with lenovo. The W series looks good, but again probably too heavy at 5.5lbs. The X1 looks to be a possibility. http://zareason.com/shop/U | 12:24 |
| kanzure | if you want light as possible then use a smartphone and a portable keyboard | 12:25 |
| archels | streety: which one just failed? | 12:26 |
| archels | I just fixed my Lenovo U430 | 12:27 |
| streety | lenovo G550, a cheap model but worked well for me for many years | 12:27 |
| streety | it still starts but windows is corrupted somehow and the CD drive isn't being picked up by the BIOS to repair/re-install | 12:28 |
| archels | haha if it has an optical drive it is definitely ripe for replacement | 12:28 |
| archels | although I have to admit I never looked into the 15"+ segment much | 12:29 |
| streety | well, DVD drive, but still true. I replaced it 3 years ago as my main computer but kept it around for those rare times when a windows machine comes in useful and as a backup | 12:31 |
| CaptHindsight | is it worth swapping in a used working G550 motherboard? | 12:33 |
| archels | .title https://wada-main-prod.s3.amazonaws.com/resources/files/wada-2015-prohibited-list-en.pdf | 12:35 |
| yoleaux | archels: Sorry, that doesn't appear to be an HTML page. | 12:35 |
| archels | indeed it isn't | 12:35 |
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| archels | The World Anti-Doping Code: The 2015 Prohibited List | 12:36 |
| streety | for what I have been using it for it wouldn't be worth the effort. If my main computer was humming along nicely I would just put it up on a shelf and not even consider taking any further action | 12:38 |
| streety | only considering buying a new laptop because my principal laptop is less than 100% as well | 12:38 |
| ParahSailin_ | asus zenbook is ok, just got one of those | 12:42 |
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| archels | ParahSailin_: what OS are you running? | 12:48 |
| ParahSailin_ | win 8.1 | 12:48 |
| archels | argh Asus is doing that power-button-in-the-corner-of-keyboard thing now as well | 12:49 |
| ParahSailin_ | ive forgotten where a power button is supposed to go | 12:50 |
| streety | looks interesting, thanks | 12:51 |
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| archels | https://en.wikipedia.org/wiki/Born_alive_laws_in_the_United_States | 12:59 |
| ParahSailin_ | what the hell is misprision | 13:01 |
| archels | .ety misprision | 13:17 |
| yoleaux | misprision (n.): ""wrong action, a failure on the part of authority," early 15c., from Anglo-French mesprisioun "mistake, error, wrong action or speech," from Old French mesprision "mistake, wrongdoing, fault, blame, crime," from mespris, past participle of …" — http://etymonline.com/index.php?term=misprision | 13:17 |
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| fenn | "a member of the species Homo sapiens, at any stage of development, who is carried in the womb" | 13:34 |
| fenn | so we can experiment all we like with superbabies right? | 13:34 |
| drethelin | heh | 13:34 |
| fenn | to say nothing of artificial wombs | 13:35 |
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| FourFire | fenn, I like the cut of your jib: "so we can experiment all we like with superbabies right?" | 14:24 |
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| mgin | yo | 14:24 |
| fenn | sup | 14:24 |
| mgin | anyone here trying to achieve immortality? | 14:25 |
| kanzure | nah we're already dead | 14:25 |
| fenn | already achieved it... | 14:25 |
| FourFire | mgin, I'm intending on dedicating my useful healthspan to researching biological indefinite lifespan | 14:25 |
| mgin | FourFire: good man! | 14:25 |
| kanzure | FourFire: that's insane; who wants to be biological indefinitely? | 14:25 |
| FourFire | immortality is a problem for us to work on in a thousand years or so | 14:25 |
| mgin | finally someone serious | 14:25 |
| mgin | what's your strategy? | 14:26 |
| FourFire | kanzure, well I'd like to stick as close to what I know works, just with improvements until the kinks are worked out of more... transcending methods | 14:26 |
| kanzure | FourFire: i don't think you know whether biology can last indefinitely. that's not a good idea. | 14:26 |
| fenn | i'm thinking along the lines of that mutant in "total recall" that has a baby embedded in his abdomen | 14:26 |
| kanzure | mgin: http://diyhpl.us/wiki/declaration | 14:26 |
| kanzure | i seem to have missed a conversation | 14:26 |
| FourFire | mgin current plans involve brute forcing some proteins I need in probability space with some tweaked evolutionary algorithms, but I haven't made much progress towards that yet so I don't want to toot my horn much about it. | 14:27 |
| FourFire | brute forcing the genes for those proteins, I mean. | 14:27 |
| mgin | what does that mean? | 14:27 |
| mgin | you want to figure out which genes code for which proteins? why? | 14:28 |
| FourFire | kanzure, of course it can't, but it can be improved to last, say twice as long? | 14:28 |
| FourFire | that's all I need, i reckon | 14:28 |
| kanzure | okay that's not the same thing as indefinite | 14:28 |
| FourFire | the cybernetics and nanotechnology approaches will be quite far along by the 2060s | 14:28 |
| mgin | kanzure: what's that for? | 14:29 |
| FourFire | "working on" and "working towards" != reached | 14:29 |
| kanzure | mgin: it's for reading...? | 14:29 |
| kanzure | fenn: i am writing a helpwanted job ad for some chemists. what should i mention? | 14:29 |
| mgin | so i read it. what of it? | 14:29 |
| kanzure | mgin: you asked where the serious people are, then i gave you the link | 14:29 |
| mgin | so? | 14:29 |
| FourFire | kanzure, I have a low probability for Cryonics and a slightly higher probability for plastination strategies working, so that's not really an option | 14:29 |
| kanzure | mgin: so nothing, you're welcome to ignore it, although i think you would be doing yourself a disservice | 14:30 |
| FourFire | I also don't want to lose all he stuff you start losing once you reach biological 50 | 14:30 |
| mgin | you're trying to suggest this link implies that you are serious about achieving immortality? | 14:30 |
| kanzure | have you read the document? | 14:30 |
| mgin | FourFire: you want to figure out which genes code for which proteins? why? | 14:30 |
| mgin | kanzure: yes | 14:31 |
| mgin | i've seen all this stuff a thousand times, what of it? | 14:31 |
| kanzure | i think you'll have to provide better criticism than that | 14:31 |
| kanzure | something like "this is not serious because x" | 14:31 |
| mgin | how am I criticizing anything? | 14:31 |
| mgin | i'm asking you what's this in reference to | 14:31 |
| kanzure | we already established that | 14:32 |
| FourFire | mgin, no, I want to "invent" (really just find) proteins which aren't know and probably don't exist in nature which do simple, low hanging fruit things which are useful | 14:32 |
| FourFire | but very unlikely to have come about due to evolution | 14:32 |
| FourFire | aren't known* | 14:32 |
| mgin | FourFire: things like what? | 14:32 |
| FourFire | and test with simulations, animals etc. until they can be integrated in the human genome without too bad side effects | 14:33 |
| kanzure | 14:29 < kanzure> mgin: you asked where the serious people are, then i gave you the link | 14:33 |
| kanzure | this already answers your question: | 14:33 |
| kanzure | 14:31 < mgin> i'm asking you what's this in reference to | 14:33 |
| mgin | [05:32.30] <mgin> you're trying to suggest this link implies that you are serious about achieving immortality? | 14:33 |
| FourFire | I'm only going to realistically work on a tiny aspect of one subproblem, but I'm betting on people like me who are already working on other aspects of the same and different problems at least being partially successful | 14:33 |
| kanzure | mgin: i don't think that's a fair question | 14:34 |
| kanzure | FourFire: you should just use subunits to make structures, problem solved, no protein folding needed | 14:34 |
| mgin | how is it fair or unfair? it's just a question. is that what you're trying to do? you're saying you are serious about achieiving immortality? i don't know, hence the question | 14:34 |
| mgin | my follow up question is "how?", if you are | 14:34 |
| kanzure | questions can be fair or unfair based on their context. i gave you a link, and now you're interrogating me about my beliefs about immortality. | 14:35 |
| FourFire | mgin, well for my part, mechanism for increased time before cellular asphyxiation, mechanism to increase physical information robustness of the genome by at least factor of Three | 14:35 |
| FourFire | those are the two main goals ATM, with a preference for the second | 14:35 |
| kanzure | mgin: i think that the ideas expressed on that link are much more serious than the rest of the immortality literature out there | 14:36 |
| FourFire | but I have ideas (and not much more) for further potential "upgrades" | 14:36 |
| kanzure | i have nfc why you would jump so weirdly with reasoning to such a distant conclusion | 14:36 |
| FourFire | (of course by the time I get around to developing someone else will have built something much better with nanotech or just good enough robotics) | 14:36 |
| mgin | FourFire: so you're trying to generate proteins which help with that? which could potentially be synthesized? | 14:36 |
| FourFire | I'm trying to find the genes, not just the proteins | 14:37 |
| mgin | what genes? | 14:37 |
| FourFire | and integrate into the genome, so that they are "naturallY" synthesized | 14:37 |
| mgin | oh | 14:37 |
| FourFire | I want to discover robust technology | 14:37 |
| FourFire | not silly elven fairytale magic which fails the instant the power goes out | 14:38 |
| mgin | so how do you know which proteins need to be synthesized? | 14:38 |
| FourFire | I'd like my enhancements to remain at least partially useful even in a loss of civilization scenario | 14:38 |
| FourFire | mgin, I don't, I need to find them, all I know is what I need the to do | 14:39 |
| FourFire | this is where the evolutionary algorithm comes in | 14:39 |
| mgin | what do they need to do? | 14:39 |
| FourFire | and I'd prefer not to talk much more about it since I haven't really made much progress so far | 14:39 |
| FourFire | PM? | 14:39 |
| mgin | well i'm just wondering generally | 14:42 |
| mgin | how do your two goals fit into life extension research? | 14:42 |
| mgin | do you think they will have significant impact on aging? | 14:42 |
| mgin | i mean, "mechanism for increased time before cellular asphyxiation, mechanism to increase physical information robustness of the genome by at least factor of Three" | 14:43 |
| FourFire | mgin, well one | 14:43 |
| FourFire | the first method: delay mechanism for cellular asphyxiation would improve healthspan outcomes for metabolism degeneration, extend it basically, and also decrease sudden death incidence (because if you take 20 minutes to drown/die of blood loss/take permanent brain damage from stroke, heart attacks etc. then someone could rescue you in time) | 14:45 |
| FourFire | the second mechanism, if it works, would allow you to retain the complete information of your genome in stem cells (where it's important) because rate of mutation from all non-nucleus-destructive sources would be orders of magnitude rarer | 14:47 |
| mgin | why does that matter? | 14:48 |
| FourFire | mgin, why are mutations bad? | 14:48 |
| FourFire | (and don't give me crap about "muh evolutions will stahp") | 14:48 |
| mgin | so your approach is to identify individual proteins which will somehow have these beneficial effects, and the genes which code for them, and then to what, insert these genes into the DNA of cells somehow so that they will get expressed? | 14:49 |
| FourFire | maybe not individuals proteins, possibly tertiary or even Quaternary structures in one mechanism | 14:51 |
| FourFire | whatever works and deson't break something else inside the cell | 14:51 |
| FourFire | I don't need something perfect on the first try, just something that works better than my base biology | 14:51 |
| mgin | how are you going to go about doing all of this? | 14:52 |
| FourFire | mgin, yes though other people are working on the gene insertion technology right now | 14:52 |
| FourFire | lots of computers, and simulations | 14:52 |
| mgin | simulating what? | 14:52 |
| FourFire | a freaking buttonne of computational power, the likes of which doesn't currently exist on this planet, but which will be available in the near decades | 14:53 |
| FourFire | Running an evolutionary algorithm which selects against arbitrary amino acid sequences and their fitness against a specific fitness function | 14:53 |
| FourFire | recombining the best 10 (or possibly 100) out of generations somewhere between 10⁵ and 10⁷ in size | 14:54 |
| mgin | what fitness function? | 14:55 |
| FourFire | recombining he best candidates into the next ~10⁵-10⁷ camndidates | 14:55 |
| FourFire | mgin I haven't written it yet, which is why frankly it's embarrassing for me to have explained this much already, for the DNA mechanism, see how well it can cross check the same basepair triplett against three sets of the same chromosome and then replace one which doesn't match if only one doesn't match, with the one the other two are | 14:57 |
| FourFire | then slide down the three chromsome copies three baspairs and repeat | 14:57 |
| FourFire | for the asphyxiation delay mechanism, 1, an organelle which can contain potentially highly reactive oxygen rich molecules, 2, a protein + chemical mechanism which, depending on environmental acidity outside the organelle membrane either, integrates O² molecules into a more stable but still fairly dense molecule which stores the oxygen (while requring a capped amount of chemical energy to decompose), OR, reverses the process | 15:01 |
| FourFire | unlocking O² molecules and letting them into the cell for metabolic use | 15:01 |
| mgin | so the premise is that we are developing the ability to insert genes into the genome, so you want to start figuring out what genes it would be helpful to add | 15:01 |
| FourFire | of course that last one is rather more ambitious and not quite as useful as the first, which is why I'm preferring to focus on it | 15:01 |
| FourFire | on the first, seems like more low hanging | 15:02 |
| FourFire | mgin, we have developed, look into CRISPR | 15:02 |
| mgin | sure | 15:02 |
| mgin | but how the hell do you simulate something like that? | 15:02 |
| FourFire | it's sorta unreliable, but it works to some extent and it's only going to be improved upon | 15:02 |
| FourFire | well I've not gotten that far yet | 15:02 |
| FourFire | but my intentions are not to simulate an entire cell, ala Wholecell.stanford.edu | 15:03 |
| FourFire | just one small area of the environment | 15:03 |
| mgin | so you're trying to figure out how to build some complex protein structure which checks DNA for anomalies and replaces them? | 15:05 |
| FourFire | and when I begin to get highly functional candidates for my molecules, I'll add the the fitness function that it must not react with x,y,z,a,b,c,fiz,buz molecules, and add one or to of each of the molecules inside the human cell to the simulation | 15:05 |
| FourFire | but by the time I'm that far along, computers I will have access to will be so much more powerful | 15:05 |
| FourFire | mgin, yep | 15:05 |
| FourFire | and I hope, that if it can overcome, with the help of native dna repair mechanism, single and double strand breaks, it will only fail in two situations | 15:06 |
| FourFire | > each of the three copies of the genome have different triplets at the given spot | 15:06 |
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| FourFire | > two of the three copies are mutated to the same different triplet | 15:07 |
| FourFire | in the first scenario, the protein should just pass on, that's an incorrectable data loss | 15:07 |
| FourFire | a silent hard error, if you like | 15:07 |
| FourFire | in the second, that's a bit flip | 15:08 |
| FourFire | and again, that's fine, reducing mutation rate by several orders of magnitude is Good enough for me | 15:08 |
| mgin | and you plan to do this with simulated proteins? can we even simulate proteins? | 15:10 |
| FourFire | well yes and no | 15:10 |
| FourFire | depends what detail of resolution you want, how many frames per nanoecond and whatnot | 15:11 |
| mgin | so we can approximately simulate them? | 15:11 |
| FourFire | all stuff I don't know the requiremnts for yet, but I'm going to be working on it for the next five years at least to get my proof of concept working | 15:11 |
| FourFire | (I intend to evolve some form of the human histone proteins without actually inputting any specific basepair data besides the apporximate length of the DNA sequence which encodes for it | 15:13 |
| FourFire | ) | 15:13 |
| kanzure | fenn: plz check http://diyhpl.us/~bryan/papers2/DNA/chemistry-hiring-ad.txt | 15:14 |
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| FourFire | if I succeed and the artificially evolved histones work as well, or possibly even better than human ones, then the method works and I won't be wasting the next decades of my life | 15:14 |
| FourFire | if not, well I spent 5 years doing science related stuff instead of becoming a politician or a stock broker or something | 15:15 |
| FourFire | and then I can reevaluate my life goals and jump on the next most likely looking approach for me to help | 15:16 |
| mgin | well i'm just asking how this works, you're saying it's possible to approximately simulate proteins? | 15:16 |
| FourFire | mgin yes, I'm using a program called GROMACS, and a visualization tool called PyMOL | 15:16 |
| FourFire | both open source, both on linux (of course) | 15:16 |
| FourFire | mgin yes, but I, personally, don't yet know what degree of precision is required of the simulation in order to get "correct" answers to interaction questions | 15:17 |
| FourFire | frankly the complexity of things like chaperonin scare me | 15:18 |
| FourFire | but the way I see it, it's like the Human genome project | 15:18 |
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| mgin | yeah i imagine proteins are insanely complex, let alone trying to simulate their behavior.. | 15:18 |
| FourFire | when they started it was literally an impossible task | 15:18 |
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| kanzure | it was hardly impossible, venter was already half-way done | 15:18 |
| fenn | i read the ad but i don't have any insight | 15:19 |
| kanzure | fenn: so "not completely shit"? | 15:19 |
| kanzure | fenn: what would you want out of a chemistry person in here? | 15:19 |
| fenn | "Hourly OK, milestone payments OK, other schedules OK. Upfront payments also OK" might be confusing, "so what exactly am i being paid to do???" | 15:19 |
| mgin | i think your approach generally makes sense at least, gene therapy is starting to become feasible... so if we can figure out which genes to code for to help enhance biology / solve aging problems, that could be a big help | 15:20 |
| kanzure | mgin: using gromacs would be a big help why? | 15:20 |
| kanzure | fenn: maybe i should just make up some arbitrary goal ("a document") and demand that they produce it....? | 15:20 |
| fenn | YOU MUST ... write this | 15:21 |
| fenn | haha | 15:21 |
| mgin | it's a pretty hard problem though... we need to know which proteins will have beneficial effects, and how to code for them genetically | 15:21 |
| fenn | personally i would like to know more about the safety of phosphoramidite nucleotides | 15:22 |
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| kanzure | there's nothing like exposing people to the harsh love of reality, so maybe the goal should be "a working oligonucleotide synthesis, either from your premises or ours" | 15:22 |
| QuadIngi | but the technology improved, and after a few years, it was only a year of work until they were done | 15:22 |
| QuadIngi | did I miss anything? | 15:22 |
| fenn | do they cause cancer, how terrible, etc | 15:22 |
| QuadIngi | last I saw is xx:21:24 | 15:22 |
| mgin | FourFire: even if you figure out a helpful protein, how do you figure out the genetic sequence to synthesize it? | 15:23 |
| kanzure | also i was considering something like just a "collaborators wanted" ad. instead of paying anyone. i mean paying people is good, but i think there might be one or two individuals that would be willing to poke their heads in here anyway without pay. | 15:23 |
| QuadIngi | mgin, ah that's the question | 15:23 |
| kanzure | mgin: you can use something called reverse transcriptase. do you even biology? :-) | 15:23 |
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| fenn | yes paying people tends to make things awkward | 15:23 |
| QuadIngi | I'm searching probability space for genes, not proteins | 15:23 |
| kanzure | wait, er | 15:23 |
| kanzure | not reverse transcriptase | 15:23 |
| QuadIngi | and I'm rating the fitness of the genes, but how the proteins they synthesize perform | 15:23 |
| fenn | protein sequencer | 15:23 |
| kanzure | what's the one for .. yes, protein sequencing, sure. | 15:23 |
| QuadIngi | s/but/by | 15:24 |
| mgin | how do you know what proteins are generated by a given gene sequence? | 15:24 |
| kanzure | mgin: you don't care; you start with the protein and then sequence the protein. | 15:24 |
| QuadIngi | it's a relatively simple program to write | 15:24 |
| mgin | oh | 15:24 |
| fenn | it's pretty straightforward to translate DNA sequence to the protein it produces, there's a small lookup table | 15:24 |
| QuadIngi | so simple the even I that can't code could write it in speudocode | 15:24 |
| fenn | .wik code of life | 15:24 |
| yoleaux | fenn: Sorry, that command (.wik) crashed. | 15:24 |
| fenn | .wik codon | 15:24 |
| yoleaux | "The genetic code is the set of rules by which information encoded within genetic material (DNA or mRNA sequences) is translated into proteins by living cells." — https://en.wikipedia.org/wiki/Codon | 15:24 |
| mgin | i know genes code for amino acids right | 15:25 |
| kanzure | -_- | 15:25 |
| mgin | er | 15:25 |
| mgin | right? | 15:25 |
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| fenn | genes code for gene products :P | 15:25 |
| fenn | biology is messy | 15:25 |
| fenn | the simplified version is that one gene makes one protein from one strand of dna | 15:26 |
| mgin | i'm just asking, how well do we really understand the mapping between genes and proteins? | 15:26 |
| fenn | er, and the gene is the one strand of dna | 15:26 |
| FourFire | fenn, which is another thing I'm uncertain about, do I reduce the search space by searching for genes, or do I increase it? | 15:26 |
| kanzure | mgin: are you asking about protein folding, or are you asking about the "central dogma"? | 15:26 |
| mgin | right, isn't protein folding a huge problem by itsefl?? | 15:26 |
| kanzure | you didn't answer my question | 15:26 |
| mgin | in other words you code for XYZ amino acids but how do you know what protein is the result | 15:27 |
| FourFire | because I imagine the the kinds of mechanisms required to do the stuff I want requires more than one part (so tertiary structures at least) | 15:27 |
| kanzure | mgin: you look up what protein it made last time, then you know | 15:27 |
| kanzure | although amino acids don't always fold into the same proteins in all conditions | 15:27 |
| mgin | that's what i'm saying, you would have to try it experimentally, that's not something we can simulate is it? | 15:27 |
| FourFire | gtg | 15:28 |
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| mgin | that seems like a huge gap in his approach, no? | 15:28 |
| kanzure | .wik central dogma of molecular biology | 15:28 |
| yoleaux | "The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system. It was first stated by Francis Crick in 1956 and re-stated in a Nature paper published in 1970:" — https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology | 15:28 |
| kanzure | mgin: yes, but previously he wasn't doing anything at all. i think this is his first project. | 15:29 |
| kanzure | and i haven't had the heart to stop him yet | 15:30 |
| kanzure | fenn: i'm afriad the ideological "OPEN-SOURCE ALL THE THINGS" or "HACK THE PLANET" "help wanted" ad wont go over well. | 15:31 |
| kanzure | maybe i should just pose it as an "academic credit" thing...... but then that will attract people that care about academic credit. | 15:31 |
| kanzure | which i'm not sure i want to deal with | 15:32 |
| kanzure | "The team also calculated the planet's equivalent of computing power: the speed of DNA transcription. Given the average rate of genetic transcription for different organismal groups, they found that the biosphere processes more than 10^24 subunits of DNA per second." | 15:33 |
| mgin | did anybody see that article on turning bacteria into computing components? that shit is fascinating to me | 15:35 |
| mgin | imagine if we could just grow a massive computer out of bacteria in a petri dish | 15:36 |
| kanzure | it would be the slowest computer ever | 15:36 |
| kanzure | there are much more interesting things to do with bacteria in a petri dish | 15:36 |
| kanzure | like nanotechnology | 15:36 |
| kanzure | https://github.com/kanzure/nanoengineer | 15:36 |
| mgin | it wouldn't necessarily be slow | 15:36 |
| mgin | what's this? | 15:37 |
| kanzure | nanotech stuff | 15:37 |
| kanzure | instead of using diamondoid mechanosynthesis you could use ribosomes to construct fusion proteins and protein subunits that form atomically-precise mechanical machinery | 15:37 |
| fenn | " Jacques Monod pointed out to me that I did not appear to understand the correct use of the word dogma, which is a belief that cannot be doubted. I did apprehend this in a vague sort of way but since I thought that all religious beliefs were without foundation, I used the word the way I myself thought about it, not as most of the world does ... that a dogma was an idea for which there was no | 15:37 |
| kanzure | it was an example of what to do with bacteria in a petri dish | 15:37 |
| mgin | this looks legit | 15:37 |
| fenn | reasonable evidence. You see!?" | 15:37 |
| mgin | holy shit | 15:37 |
| fenn | what does nanoengineer have to do with bacteria in a petri dish? | 15:38 |
| kanzure | i was giving him examples of molecular things to build with bacteria | 15:39 |
| kanzure | he wanted to do computation instead | 15:39 |
| mgin | i've seen this nanofactory video many times over the years | 15:39 |
| kanzure | yes, well, now you know who made it | 15:39 |
| mgin | and the animations of molecular machines | 15:39 |
| mgin | you really made this stuff? | 15:39 |
| fenn | lol | 15:39 |
| kanzure | the original developers of nanoengineer were responsible for the nanofactory animations | 15:39 |
| kanzure | the README specifically says this........ | 15:40 |
| kanzure | (although the nanoengineer devs did not make the animation itself, they were involved/responsible/culpable) | 15:40 |
| fenn | kanzure was saddled with upkeep and archival of a dead project | 15:40 |
| kanzure | ((the foresight challenge)) | 15:40 |
| mgin | oh | 15:41 |
| kanzure | .wa mass of adenosine in grmas | 15:41 |
| kanzure | .wa mass of adenosine in grams | 15:41 |
| yoleaux | convert adenosine: molar mass to grams: 267.241 g/mol (grams per mole); Additional conversion: 0.267241 kg/mol (kilograms per mole); Comparisons: ~0.37 × molar mass of fullerene (~721 g/mol); ~1.4 × molar mass of caffeine (~194 g/mol); ~4.6 × molar mass of sodium chloride (~58 g/mol); Corresponding quantities: Mass of a molecule m from m = M/N_A:: 4.4×10⁻²² grams: 4.4×10⁻²⁵ kg (kilograms): 267 u (unified atomic mass units | 15:41 |
| yoleaux | ): 267 daltons | 15:41 |
| yoleaux | convert adenosine: molar mass to grams: 267.241 g/mol (grams per mole); Additional conversion: 0.267241 kg/mol (kilograms per mole); Comparisons: ~0.37 × molar mass of fullerene (~721 g/mol); ~1.4 × molar mass of caffeine (~194 g/mol); ~4.6 × molar mass of sodium chloride (~58 g/mol); Corresponding quantities: Mass of a molecule m from m = M/N_A:: 4.4×10⁻²² grams: 4.4×10⁻²⁵ kg (kilograms): 267 u (unified atomic mass units | 15:41 |
| yoleaux | ): 267 daltons | 15:41 |
| fenn | there's nothing wrong with the software really, but they never really developed a user base | 15:41 |
| kanzure | .wa 4*(10^(-25)) grams * (10^24 / second) | 15:42 |
| fenn | i wish wolfram alpha would just shut the fuck up and give you the answer you asked for instead of all this other crap | 15:42 |
| yoleaux | 4×10⁻²⁵ grams×10²⁴/(second): 0.4 g/s (grams per second); Unit conversions: 4×10⁻⁴ kg/s (kilograms per second); 24 g/min (grams per minute); 0.024 kg/min (kilograms per minute); 24000 mg/min (milligrams per minute); 2.4×10⁷ µg/min (micrograms per minute); Basic unit dimensions: [mass] [time]⁽⁻¹⁾ | 15:42 |
| mgin | so | 15:42 |
| kanzure | the whole biosphere is only making 0.4 grams/second of nucleotides?? | 15:42 |
| mgin | how is this sort of nanoscale modeling useful? | 15:43 |
| kanzure | wait shouldn't this be exponential or something | 15:43 |
| mgin | sounds like it isn't given that it's a dead project with no user base | 15:43 |
| kanzure | why is tihs a linear rate >:( | 15:43 |
| fenn | is 10^-25 supposed to be avogadro's number? | 15:43 |
| kanzure | oh it said kilograms | 15:44 |
| kanzure | .wa 4*(10^(-25)) kilograms * (10^24 / second) | 15:44 |
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| yoleaux | 4×10⁻²⁵ kg (kilograms)×10²⁴/(second): 0.4 kg/s (kilograms per second); Unit conversions: 400 g/s (grams per second); 24 kg/min (kilograms per minute); 24000 g/min (grams per minute); 2.4×10⁷ mg/min (milligrams per minute); 2.4×10¹⁰ µg/min (micrograms per minute); Basic unit dimensions: [mass] [time]⁽⁻¹⁾ | 15:44 |
| kanzure | 0.4 kg/sec sounds better | 15:44 |
| kanzure | although not really. hrm. | 15:44 |
| kanzure | perhaps i should do this the other way around: what would be a reasonable guestimate for the mass per second of dna being produced by the biosphere? | 15:45 |
| fenn | "subunit" is a vague word | 15:46 |
| kanzure | i think they meant nucleotide | 15:46 |
| kanzure | http://www.nytimes.com/2015/07/21/science/counting-all-the-dna-on-earth.html | 15:47 |
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| kanzure | yashgaroth: yo | 15:48 |
| yashgaroth | sup | 15:48 |
| kanzure | stuff | 15:48 |
| kanzure | you? | 15:48 |
| yashgaroth | same | 15:48 |
| fenn | yo/sup protocol completed | 15:48 |
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| kanzure | yashgaroth: we have someone willing to build an inkjet dna synthesizer, but we need a chemist i think | 15:49 |
| fenn | begin transmitting data on port 698896 | 15:49 |
| yashgaroth | ah was about to ask: so rather than read through a megabyte of electrowetting, how's the new dna synth project going? | 15:49 |
| yashgaroth | yes a chemist would be the missing link in the group | 15:49 |
| kanzure | yashgaroth: also for gene assembly we are thinking of a cycling ligase reaction on an electrowetting surface for moving and merging droplets (although it's possible that this system would also be used for phosphoramidite chemistry too...?) | 15:49 |
| kanzure | "cycled ligation assembly" http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107329 | 15:50 |
| yashgaroth | fine I'll find out what electrowetting is | 15:50 |
| kanzure | hydrophobic drops on a surface and then electrodes under the thin surface, electrodes cause drops to move | 15:50 |
| fenn | electrowetting is just moving tiny drops around on a slide | 15:50 |
| yashgaroth | ok cool do that | 15:51 |
| fenn | here's a dumb video http://youtu.be/jpbUvSyeRg0 | 15:52 |
| fenn | .title | 15:52 |
| yoleaux | Droplet Dispensing and Mixing in Digital Microfluidics - YouTube | 15:52 |
| yashgaroth | that linked article still requires rather large pieces of dna though | 15:52 |
| kanzure | 40 bp? | 15:52 |
| yashgaroth | isn't that just for the linker bit? lemme see | 15:53 |
| kanzure | yes, but i don't know if they tested the requirement of length for the non-linker strands | 15:53 |
| kanzure | "The efficiency of CLAs decreases as the size of the assembled DNA fragments grows above 5–6 kb" | 15:54 |
| kanzure | "This effect is likely due to the probabilistic nature of CLAs" (so can be fixed with heat and time and stuff) | 15:54 |
| yashgaroth | they become far less likely to randomly orient and line up and behave yeah | 15:54 |
| fenn | i was thinking the first ligation reaction would be all linkers (50 bp oligos say) that overlapped by half | 15:54 |
| kanzure | "At the scale of the most common DNA assembly goals, using fragments from <500 bp to 5 kb, cycled-ligation assemblies are efficient" | 15:55 |
| kanzure | doesn't say how much smaller than 500 bp | 15:55 |
| yashgaroth | I really should just order a bunch of t4 ligase and 10bp oligos and test that out before I try to preach it any more | 15:56 |
| fenn | 10 is pretty short | 15:56 |
| kanzure | ok well let me know what the total amounts to | 15:56 |
| yashgaroth | 10's a lot better than we were planning to work with, when a million seemed too high for microplates | 15:57 |
| kanzure | have you ever done emulsion pcr? | 15:57 |
| fenn | oh well 10 is not much larger than 6 | 15:57 |
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| yashgaroth | I avoid pcr whenever possible, and no | 15:57 |
| fenn | why dont you like pcr? | 15:58 |
| yashgaroth | biology's already enough hopes and guesses and frustration without pcr | 15:58 |
| yashgaroth | also I mostly do protein work these days, lot fewer shrines at the sds-page bench than a pcr station | 15:59 |
| fenn | so you dont have good luck with primer design? | 15:59 |
| yashgaroth | no I'm great at that shit, usually it works for me, pcr is just a pain | 15:59 |
| yashgaroth | just because I dislike something doesn't mean I'm not good at it | 16:00 |
| kanzure | thermocyclers should do pcr prep | 16:00 |
| yashgaroth | anyway! iirc 10 was much closer to being feasible for t4 ligase recognition than 6, will try and find some data | 16:01 |
| fenn | do you think it would work if there were a lot of other nicks along the strand | 16:01 |
| kanzure | fenn: i would worry about spot density in the electrowetting approach. | 16:02 |
| yashgaroth | how many nicks we talking here | 16:03 |
| fenn | like if you had a strand made entirely of 10bp oligos overlapping by 5bp each | 16:03 |
| fenn | or 6bp oligos overlapping by 3 | 16:03 |
| yashgaroth | well the hope was to do them one at a time, with that many nicks it'd fall apart if you did them all at once | 16:03 |
| kanzure | if you overlap by 3 on both ends then you have no unique content | 16:03 |
| fenn | it falls apart because there is more force tugging on the longer strand? | 16:06 |
| yashgaroth | just a long-ish strand composed of overlapping 10mers seems unstable, and the more you add at once the higher chance that you get unwanted combination between the wrong strands | 16:07 |
| yashgaroth | possibly you could add a few oligos into the mixture at a time, depending on their sequences, if that'd help speed it up | 16:08 |
| yashgaroth | and then there was something about washing in between 10mers just so trace amts. of previous oligos don't hang around and fuck their way into the chain later on | 16:09 |
| kanzure | hmm a wash step with electrowetting. or even with the flood group method. uhh... | 16:10 |
| kanzure | if we were using magnetic beads then we could turn on a magnet and then blast away all the surrounding liquids | 16:11 |
| kanzure | but i dunno if beads could be transported in the droplets | 16:11 |
| kanzure | .title https://www.youtube.com/watch?v=P-LsWmy6hqo | 16:12 |
| yoleaux | Concentration of polystyrene beads - YouTube | 16:12 |
| yashgaroth | another fun project would be to investigate salt/pH/temp conditions that improve the ligation accuracy/efficiency/speed for this, maybe apply the inkjetted array approach somehow | 16:13 |
| fenn | yashgaroth: what's a good way of removing everything but the longest strand in a droplet that doesn't involve beads? | 16:14 |
| * fenn sick of hearing about beads | 16:14 | |
| yashgaroth | droplet? um feed it into a tiny capillary gel and electrophorese the smaller stuff out maybe | 16:14 |
| fenn | that was my first thought but it seemed complicated | 16:15 |
| yashgaroth | well when your choice is between complicated and impossible... | 16:15 |
| fenn | alternatively remove only the longest strand and come back for it later | 16:15 |
| yashgaroth | incomprehensibly small ultracentrifuge | 16:15 |
| fenn | or some sort of process that causes longer strands to precipitate faster than shorter strands do | 16:15 |
| yashgaroth | what's the g-force on atp synthase? | 16:16 |
| fenn | heh | 16:16 |
| fenn | it's actually more like a crankshaft than a turbine | 16:16 |
| yashgaroth | a guy can dream | 16:16 |
| yashgaroth | the precipitation one is a possibility, depends how absolutely selective you need to be | 16:17 |
| fenn | just to get rid of accumulating side products and mismatched/malformed oligos | 16:18 |
| kanzure | other than beads and linking to a surface, and complex gel/hplc shit that nobody wants, i'm not sure how to not wash away oligos | 16:18 |
| fenn | hm i should clean my desk | 16:20 |
| yashgaroth | yeah I always assumed they'd be attached to a surface | 16:20 |
| kanzure | they can be attached to a surface (and not a bead), but then moving droplets is not as useful | 16:20 |
| yashgaroth | wait are we moving the chain between places on the oligo board? I thought it was stationary chain and then you droplet the oligos onto it | 16:21 |
| kanzure | uh.. undecided i think? | 16:22 |
| fenn | it's a lot simpler to move the oligos around: http://fennetic.net/irc/dna_printer_EWOD_oligo_resuspension_linear.png | 16:23 |
| kanzure | i thought both were moving around | 16:23 |
| fenn | er, uh, terminology fail | 16:23 |
| fenn | is there a word for a combination of synthesized short strands that is slightly longer? | 16:23 |
| kanzure | "synthesized oligos (bound)" how are they going to be ligated to anything if everything prior in the path was also tied down...? | 16:23 |
| kanzure | oh, cleaved | 16:24 |
| fenn | that's why the droplet also contains a cleaving enzyme | 16:24 |
| kanzure | ok. so everything gets cleaved. | 16:24 |
| fenn | yes | 16:24 |
| yashgaroth | cleaving or nicking? | 16:25 |
| fenn | the synthesized oligos are single stranded | 16:25 |
| yashgaroth | yes | 16:25 |
| yashgaroth | .title http://www.biomedcentral.com/content/pdf/1756-0500-3-291.pdf | 16:27 |
| yoleaux | yashgaroth: Sorry, that doesn't appear to be an HTML page. | 16:27 |
| yashgaroth | fuck you yoleaux | 16:27 |
| yashgaroth | .title http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994885/ | 16:28 |
| yoleaux | Efficient assembly of very short oligonucleotides using T4 DNA Ligase | 16:28 |
| yashgaroth | decent success with 8mers | 16:28 |
| kanzure | "From the ligation experiments, it was concluded that DNA synthesis was feasible with octamers. To improve the pace of such a method, a hierarchical approach was designed in which multiple intermediate fragments could be constructed from octamers in parallel and then combined in a repeated pair-wise manner. The solid-support used to anchor growing intermediate fragments was designed such that digestion with BbsI would release any ... | 16:30 |
| kanzure | ... attached fragment while retaining a 4-nt overhang (Figure (Figure3a).3a). Released intermediates could then be used in further ligations. Four distinct bead sets were created each with a unique 4-nt overhang (Figure (Figure3b).3b). The overhangs for the solid support adaptors were constructed to be complementary to evenly distributed regions of the 128-bp target such that eight octamers, overlapping in 4-nt frame shifts, would ... | 16:30 |
| kanzure | ... tile between each region." | 16:30 |
| kanzure | figure 3 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994885/bin/1756-0500-3-291-3.jpg | 16:30 |
| kanzure | or er, better figure 3 link http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994885/figure/F3/ | 16:30 |
| kanzure | hm. | 16:33 |
| kanzure | "Octamers that are complete palindromes will self-hybridize, and those with 4-nt palindromic ends will result in self-polymerization. Further, repetitive sequences longer than 8 bp cannot be synthesized from octamers, and cleavage from the solid support by BbsI digestions requires that the recognition site for this enzyme not be encoded elsewhere within the assembled sequence" | 16:35 |
| kanzure | trying to figure out why no repetitive sequences | 16:36 |
| kanzure | "In principle, it is also possible to include in the design longer, custom oligos that span problematic sequences, although at increased cost" well ok | 16:36 |
| kanzure | yashgaroth: so no more electroporation plans? or did i misinterpret | 16:40 |
| yashgaroth | pretty much for now yeah | 16:40 |
| yashgaroth | rather high number of injections with a specialized device, probably not much mass appeal there | 16:41 |
| kanzure | not with that attitude | 16:41 |
| kanzure | did you see the cambrian genomics pics yesterday? | 16:41 |
| yashgaroth | a couple of 'em I think, wanna get on that ebay firesale | 16:42 |
| yashgaroth | also if I can get like 100 mg/L of the protein in pichia instead, it should be cost effective, I mean I haven't worked out the break-even point but we'll see | 16:44 |
| yashgaroth | & some people have been having success with AAV therapy, I never looked into it since you need a clinic for the immunosuppression, but said people have that and it's supposedly working for them | 16:45 |
| streety | what's the protein? | 16:45 |
| yashgaroth | follistatin, a myostatin inhibitor, fusion to an antibody Fc region | 16:46 |
| yashgaroth | err, fused* | 16:47 |
| streety | whats the reason for the Fc fusion? I've seen it done elsewhere but never knew why | 16:47 |
| yashgaroth | extends the serum half-life from 1 hour to 2 weeks, roughly | 16:48 |
| yashgaroth | also makes it somewhat easier to purify, but mostly it's for the half-life | 16:48 |
| streety | that is quite an improvement, thanks | 16:49 |
| yashgaroth | and ideally it aids clearance of the target protein; so the follistatin-Fc binds the circulating myostatin, then that whole complex gets pinocytosed into cells, vesicle acidifies causing complex to separate, Fc-fusion protein gets recycled outside the cell by FcRn receptor, myostatin gets digested | 16:51 |
| kanzure | ewod with microelectrodes (although the spacing seems to be 1 millimeter... er..) http://ir.nctu.edu.tw/bitstream/11536/15046/1/000298136700010.pdf | 16:59 |
| kanzure | ah right, there was the picoliter ewod paper http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613134/ | 17:01 |
| kanzure | magnetic bead trapping seems to work, it's used in lots of papers and it seems to just require a giant magnet under the device | 17:08 |
| kanzure | not sure why fenn doesn't like this | 17:08 |
| fenn | SCOEW looks really cool http://teitell-lab.com/wp-content/uploads/2014/02/2010_4_Park.pdf | 17:09 |
| kanzure | "Dynal® MyOne™ Streptavidin magnetic beads (1.05 µm diameter)" | 17:09 |
| fenn | i'm just tired of thinking about beads i guess | 17:10 |
| fenn | it's really hot today | 17:12 |
| fenn | we just had a brownout | 17:13 |
| kanzure | i think i would die if texas had brownouts | 17:13 |
| kanzure | path coordination planner thingy for scoew-stuff http://webpages.uncc.edu/sakella/papers/MaAkellaICRA12.pdf | 17:14 |
| fenn | obviously they should swarm like boids | 17:16 |
| fenn | each droplet should be incentivized as a purely rational economic actor to improve its market value | 17:16 |
| kanzure | there will probably be some % failure, so plans should have easy degradations available when there is a merge failure or w/e | 17:17 |
| kanzure | here is one that uses a projector to move droplets ftp://222.18.54.49/Papers/ICRA%202013/media/files/papers_videos/2091.pdf | 17:18 |
| kanzure | "Recent work in optically activated microfluidic devices has focused on reducing the droplet actuation voltage and optical source intensity. Pei et al. [18] reported a lightactuated droplet manipulation (LADM) device in which the optical source is a conventional data projector (DELL 4210X) instead of a laser. The aggressive scaling of the dielectric thickness in the device fabrication helps them achieve high speed droplet manipulation (2 ... | 17:19 |
| kanzure | ... cm/sec) in low optical intensity (3 W/cm^2)" | 17:19 |
| kanzure | pdf of [18] http://www.colorado.edu/engineering/MCEN/MEMSII/LiteratureReview_2010/B_Light-actuated_microfluidic_2010.pdf | 17:20 |
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| kanzure | they manipulated 5 nL droplets on a 1.65 cm^2 surface | 17:24 |
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| kanzure | not sure how to combine a magnet with the lcd/dmd approach to "virtual electrodes". magnet seems likely to be disruptive to the other components? i guess you don't need "virtual electrodes" during a wash cycle so... just move the projector away. | 17:30 |
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| kanzure | oh also i think you have to plan to wash different drops at the same time, since you can't selectively isolate individual magnetic beads or droplets or whatever. so anything that isn't being "washed" needs to stay immobile until the wash cycle is done for the participating drops/beads. | 17:31 |
| kanzure | here is how they used their projector eventually, http://nanophotonics.eecs.berkeley.edu/Publications/Conference/files/241/Pei%20et%20al.%20-%202013%20-%20Isothermal%20real-time%20polymerase%20chain%20reaction%20det.pdf | 17:32 |
| kanzure | these are 1 mm diameter droplets :-/ | 17:34 |
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| kanzure | hm he also claims to have done electroporation with this | 17:38 |
| kanzure | he included a parts list, how nice of him | 17:41 |
| fenn | i dont get how you're supposed to put thousands of samples collected from the wild on a chip, and if you're not doing large numbers then what's the point of using a chip in the first place | 17:42 |
| fenn | the herpes pcr detection paper | 17:43 |
| fenn | anyway SCOEW looked simpler | 17:44 |
| fenn | so many acronyms | 17:45 |
| kanzure | how is it simpler than projecting light? heh | 17:46 |
| fenn | well, you can just put the slide on top of an lcd screen | 17:46 |
| fenn | which is crazy but apparently it works | 17:46 |
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| fenn | but it's simpler because there's no cover slip and there are less layers to be deposited on the chip | 17:47 |
| kanzure | the thesis with the bill of materials for the projector one is http://diyhpl.us/~bryan/papers2/microfluidics/Light-induced%20electrokinetics:%20A%20path%20to%20a%20versatile%20micro%20total%20analysis%20system%20(projector)%20-%20Justin%20K.%20Valley%20-%20thesis.pdf | 17:49 |
| kanzure | "Actuation at a distance of microelectromechanical systems using photoelectrowetting: proof-of-concept" http://arxiv.org/pdf/1201.2873.pdf (i guess someone figured they should confirm this shit) | 17:52 |
| fenn | the ftp...2091.pdf paper was the same basic physics as SCOEW | 17:53 |
| fenn | why do people always have such terrible document names | 17:53 |
| fenn | "towards automated optoelectrowetting on dielectric devices for multi-axis droplet manipulation" | 17:54 |
| kanzure | the thesis compares the physics | 17:55 |
| kanzure | in more detail | 17:55 |
| kanzure | wasn't there another photo radiation result that people were surprised about recently in another context | 17:56 |
| fenn | was it firing lasers through fiber optics at beads in sequencer wells | 17:59 |
| kanzure | ah, optical lift http://gnusha.org/logs/2014-06-02.log | 17:59 |
| kanzure | followed by 15:07 < kanzure> why are people always more interested in cryonics? there are perfectly good brains still living that could continue to live | 18:01 |
| fenn | you are your own worst enemy :P | 18:01 |
| kanzure | he has a good point | 18:02 |
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| kanzure | .wik optical lift | 18:09 |
| yoleaux | "Optical lift is an optical analogue of aerodynamic lift, in which a cambered refractive object with differently shaped top and bottom surfaces experiences a stable transverse lift force when placed in a uniform stream of light." — https://en.wikipedia.org/wiki/Optical_lift | 18:09 |
| kanzure | .wik optical force | 18:09 |
| yoleaux | "The optical force is a phenomenon whereby beams of light can attract and repel each other. The force acts along an axis which is perpendicular to the light beams. Because of this, parallel beams can be induced to converge or diverge. The optical force works on a microscopic scale, and cannot currently be detected at larger scales." — https://en.wikipedia.org/wiki/Optical_force | 18:09 |
| kanzure | maybe egan was right, and everyone needs to stop doing math immediately lest we accidentally make the wrong branch of reality happen | 18:11 |
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| fenn | quintillions of bits madly whirling hard drives spinning doom approches ever closer | 18:26 |
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| juri_ | fenn: you know how to party. | 18:33 |
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| juri_ | Have we thought of using electrical charge to increase the odds of our droplets landing on top of one another? | 18:35 |
| fenn | that's not really a problem from what the captain says | 18:37 |
| juri_ | well, if we're using charge to move droplets of water around, might as well use the same equipment to help target droplets. | 18:38 |
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| CaptHindsight | I can make an electrostatic printhead | 18:58 |
| CaptHindsight | in the inkjet scenario droplets land on top of the previous material | 19:00 |
| CaptHindsight | how the droplets behave is a matter of the temperature and surface tension or both the droplet and the previous layer | 19:00 |
| CaptHindsight | or/of | 19:01 |
| CaptHindsight | since the glass slides are treated with siloxane that helps keep the initial drops from spreading | 19:02 |
| CaptHindsight | after a layer or few of bases the drops are still contained if the drops spread down the sides of the previous oligo layers | 19:04 |
| CaptHindsight | I didn't see any comments about the surface tension of the previously completed oligo layers | 19:05 |
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| kanzure | you can also have a pre-programmed surface where you etch a patter ninto the surface, such that droplets would be set on a specific program of movement thanks to surface tension | 19:54 |
| kanzure | such a pre-programmed surface tension path could also take into account things like "it takes a while to load the other droplets" (although i think you'd still have to time everything pretty well) | 19:54 |
| kanzure | i think it would also be compatible with wash steps (lock the magnetic beads to the surface with a giant magnet, wash, then put water droplets at the start, by the time they get to the place with the magnetic beads, unlock the beads, then let them continue down the path) | 19:56 |
| kanzure | surface should be on a one degree incline or slant | 19:57 |
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| kanzure | or, instead of putting droplets at the same start position to go all the way back to the beginning and then pick up the beads, you can have shortcut spots in a few places although this may negatively effect your other route geometry | 20:00 |
| kanzure | "Inkjet patterned superhydrophobic paper for open-air surface microfluidic devices" http://pubs.rsc.org/en/content/articlelanding/2014/lc/c3lc51248g | 20:08 |
| kanzure | CaptHindsight: what do you think of that one? | 20:08 |
| kanzure | this isn't droplets but here's one for "continuous flow" on paper over sharpied patterns http://repositorium.sdum.uminho.pt/bitstream/1822/24877/1/17612-artigo%20plataforma.pdf | 20:16 |
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| kanzure | some droplet math modeling of sliding a droplet over alternating stripes of hydrophilic and hydrophobic surfaces http://arxiv.org/pdf/1310.4803.pdf | 20:24 |
| kanzure | welp nevermind about the patterned surfaces thing. i thought i had seen that before but i guess i was making that up. | 20:26 |
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| kanzure | inkjet emulsion pcr http://ir.semi.ac.cn/bitstream/172111/26165/1/A%20novel%20picoliter%20droplet%20array%20for%20parallel%20real-time%20polymerase%20chain%20reaction%20based%20on%20double-inkjet%20printing%20.pdf | 20:32 |
| juri_ | you give me such interesting reading assignments. | 20:38 |
| kanzure | i think you mean "everyone else i know is boring" | 20:41 |
| juri_ | I might. ;) | 20:46 |
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| fenn | i am reading this machine tool engineering thesis again http://pergatory.mit.edu/research/Cortesi/index.html | 20:50 |
| fenn | bedtime stories | 20:50 |
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| ebowden | http://www.smbc-comics.com/comics/1437229685-20150718.png | 20:56 |
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