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| anachronick | hello... goodmorning | 04:28 |
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| JayDugger | Good morning to you. | 05:21 |
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| nmz787_w1 | I wasted an entire day because some perl script parsing/running seems to have changed and I needed to prefix a variable with the keyword "my" | 10:27 |
| chris_99 | :( | 10:27 |
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| kanzure | scope matters n' such | 10:42 |
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| nmz787_w1 | doesn't 'my' just keep it local? I was only using it locally to begin | 10:48 |
| nmz787_w1 | I can only imagine the corporate IT/sysAdmin overlords changed something in the perl version or something weird | 10:48 |
| kanzure | i wonder if we could get the one brain sleeping trick to work for humans | 10:49 |
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| kanzure | or rather, one hemisphere | 10:49 |
| chris_99 | do any land animals do that? | 10:49 |
| kanzure | some birds-- maybe some flightless birds if you're lucy | 10:51 |
| kanzure | *lucky | 10:52 |
| chris_99 | intriguing cool, i'd only heard about dolphins/whales doing it before | 10:52 |
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| kanzure | there's probably a pathogen that improves eyesight somewhere | 11:33 |
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| kanzure | also, for dna assembly protein systems under artificial selection, alternating strands of XNA and DNA should be considered so that neighboring strands are never the same molecule type | 11:35 |
| kanzure | (or GNA or something) | 11:36 |
| kanzure | i suppose the whole point of XNA was that proteins treat it almost like DNA, so perhaps that's self-defeating | 11:42 |
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| kanzure | wikipedia's articles on recombinases should really have a state transition diagram or at least a list of the states | 12:00 |
| kanzure | i don't even need the diagram part | 12:00 |
| kanzure | cas9 http://www.rcsb.org/pdb/explore/explore.do?structureId=4OO8 http://www.artofthecell.com/animation/crispr-cas9-gene-editing | 12:13 |
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| kanzure | "Recent advances in DNA assembly technologies" lice.btc.calendar.opentimestamps.org | 12:41 |
| kanzure | er... what. | 12:41 |
| kanzure | "Recent advances in DNA assembly technologies" http://www.scs.illinois.edu/~zhaogrp/publications/HZ182.pdf | 12:41 |
| kanzure | "In the in vivo homologous recombination-based assembly methods, NHEJ of the linearized vectors contributes to most of the false positives. One solution could be to introduce a counter-selective marker at the cloning site (Anderson and Haj-Ahmad, 2003). In order to survive, the host must have the counter-selective marker replaced by the designated inserts. To further minimize this problem, separatin | 12:50 |
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| kanzure | g the essential elements on the vector, ... | 12:50 |
| kanzure | ... selection marker and episome has been proposed (Kuijpers et al., 2013). As the host organism needs at least 2 NHEJs to incorporate both elements, the probability of false positives was greatly reduced. With this new scheme, nine fragments were assembled by 60-bp overlapping regions with a correct yield of 95%." | 12:50 |
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| kanzure | "Improving ancient DNA genome assembly" https://peerj.com/preprints/2383.pdf | 12:57 |
| kanzure | cc maaku | 12:57 |
| kanzure | "High molecular weight DNA assembly in vivo for synthetic biology applications" http://www.tandfonline.com/doi/full/10.3109/07388551.2016.1141394 | 13:02 |
| kanzure | "A synthetic pathway for the fixation of carbon dioxide in vitro" http://science.sciencemag.org/content/354/6314/900.full | 13:13 |
| kanzure | er, i mean http://science.sciencemag.org/content/354/6314/900 | 13:13 |
| kanzure | "Some neurotransmitters are regulated by base excision repair in neurons. My very rough calculations suggested this would wreck the DNA on average after about 100 years." | 13:22 |
| kanzure | "I mean they're actually cutting a base out and changing it to up-regulate or down-regulate a neurotransmitter, and the error rate is 1 in n-thousand | 13:23 |
| kanzure | " | 13:23 |
| kanzure | this particular neural regulation mechanism sounds stupid, and we should replace it with something less stupid | 13:24 |
| kanzure | "nobody would pick it up with normal sequencing, because the results would be different for every strand of DNA" | 13:25 |
| kanzure | here's my proposal for selection projects for DNA assembly and homologous recombination in cells https://groups.google.com/d/msg/enzymaticsynthesis/uyZqtJO24RE/lApLb4JmCAAJ | 13:28 |
| kanzure | cc yashgaroth | 13:30 |
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| kanzure | "Ex vivo DNA assembly" http://journal.frontiersin.org/article/10.3389/fbioe.2013.00012/full | 14:03 |
| kanzure | "These homology-based approaches are extremely versatile, but there are disadvantages associated with each. Although isothermal DNA assembly provides a quick and reliable way to simultaneously join together multiple pieces of DNA, the cost of the purified enzymes needed to carry out the assembly reaction is non-trivial (~US$3/reaction formulated in-house). TAR cloning in S. cerevisiae is considera | 14:03 |
| kanzure | bly cheaper but requires: (1) ... | 14:03 |
| kanzure | ... preparation of yeast spheroplasts, (2) the inclusion of both a selection marker and replication origin that function in yeast, and (3) the subsequent purification of the assembled DNA product (if the DNA is intended to be used in an organism other than yeast). This is a long process that takes 8-9 days to complete (Gibson et al., 2008)." | 14:03 |
| kanzure | "To address the monetary cost of ISO assembly and the time cost of TAR cloning, we hypothesized that the DNA repair machinery endogenous to yeast and other microorganisms would remain functional within cellular lysates and should be able to catalyze the assembly of linear and circular DNA constructs in just hours. We tested this hypothesis using cellular lysates derived from S. cerevisiae, E. coli, | 14:03 |
| kanzure | and Deinococcus radiodurans, an ... | 14:03 |
| kanzure | ... extremophilic bacterium known to have exceedingly efficient dsDNA repair capabilities." | 14:03 |
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| kanzure | "IVA cloning: A single-tube universal cloning system exploiting bacterial in vivo assembly" http://www.nature.com/articles/srep27459 | 14:12 |
| kanzure | "IVA cloning exploits a recA-independent recombination pathway, which is emerging as a powerful tool in DNA manipulation. Initial reports of this bacterial pathway and its application to cloning were not rapidly adopted, possibly due to the simultaneous reporting of in vivo cloning using bacterial strains expressing phage recombinases[51], which are now widely used for genome engineering[52]. While | 14:17 |
| kanzure | the recA-independent pathway has ... | 14:18 |
| kanzure | ... recently been utilised as a cloning tool in AQUA cloning, the protocols involved for its use in vivo require multiple PCRs, gel extraction, mixing of DNA fragments and incubation prior to transformation[28, 29, 30]. Although cost-effective when compared to enzymatic assembly methods, protocols are significantly longer (3h30 from set-up to transformation). IVA cloning is efficient without such re | 14:18 |
| kanzure | quirements, providing a fast, versatile ... | 14:18 |
| kanzure | ... and cost-effective system that outperforms all current cloning methods. [...] Although the recombination mechanism is still unknown, it is clear that in vivo DNA assembly using common bacterial strains is a powerful tool for molecular cloning." | 14:18 |
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| kanzure | irssi people implemented their own "splitlong.pl" https://github.com/irssi/irssi/pull/29 | 14:31 |
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| kanzure | as for cell lysate dna repair homologous recombination stuff, if that is true then someone should try doing chimeric optimization from multiple dna repair pathways from radiation-hardened strains ... | 15:32 |
| kanzure | that would be a good first step for optimization of that. | 15:33 |
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| kanzure | yashgaroth: https://groups.google.com/d/msg/enzymaticsynthesis/uyZqtJO24RE/lApLb4JmCAAJ | 18:57 |
| kanzure | yashgaroth: also, "in vivo assembly cloning" in single step pcr http://www.nature.com/articles/srep27459 | 19:02 |
| yashgaroth | yeah there's a lot of possibilities...enzymes to melt partially-matching ssDNAs and keep them cycling to other oligos, enzymes to stabilize matching strands at higher temperatures until they get ligated; XNAs could be useful, there's quite a few out there and they do have sometimes quite different properties to DNA | 19:03 |
| kanzure | ah wasn't aware. i only know about like 3-4 non-RNA non-DNA things | 19:04 |
| kanzure | . | 19:04 |
| yashgaroth | I'm wary of doing it in cells since they've got hundreds of other DNA-interacting proteins, it's less of an issue in e.coli than yeast but still there's a lot of unknowns | 19:04 |
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| yashgaroth | well, it goes all the way up to morpholinos and stuff, but I assume we'd want to keep them close enough to natural DNA that they can still be replicated etc | 19:04 |
| kanzure | but also, why isn't yeast homologous recombination optimized to hell and back by now for DNA assembly from small fragments? | 19:04 |
| kanzure | surely someone could sit around and find a strain of yeast that does better homologous recombination or something | 19:06 |
| yashgaroth | like, by humans? they're a bit of a pain to get DNA into, especially a bunch of different fragments, and you can't have selection on all of the fragments so you'd end up blasting them with a ton of DNA | 19:06 |
| kanzure | hm @ "you can't have selection on all of the fragments"? | 19:07 |
| yashgaroth | well normally you make sure they take up the DNA because if they don't they die to antibiotic selection, but if you've got a hundred arbitrary DNA fragments they can't all have a different selection mechanism to ensure they all got uptaken | 19:07 |
| kanzure | ah. agreed. yes, you have to work up from fewer fragments to more fragments over time/generations. :( | 19:08 |
| yashgaroth | and yeast have got both a cell wall and nuclear membrane so the efficiency sucks | 19:08 |
| kanzure | turns out people have been using ecoli for homologous recombination lately, anyway. so fuck yeast. | 19:09 |
| yashgaroth | I mean there are viral methods or similar, not sure if anyone's characterized yeast viruses but I'm sure they have viruses...or engineer the cells to pick up foreign DNA more easily | 19:09 |
| kanzure | lots of cell membrane pores for random dna uptake, sure. | 19:11 |
| kanzure | i think some of those proteins have even been characterized by now | 19:11 |
| kanzure | as for getting a copy of all the dna into each cell, that's either a problem of concentration or a problem of attaching dna to microbeads and blasting the microbeads into each cell | 19:12 |
| yashgaroth | assuming they can chop the DNA off the beads once they're inside, which there's probably an enzymatic method to do | 19:14 |
| kanzure | (you could do light lysis where the dna could be cut off the bead) | 19:14 |
| kanzure | yes you were reading my brainthinks | 19:14 |
| yashgaroth | that'd be a lot easier than enzymatic tbh | 19:14 |
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| yashgaroth | if you put XNAs and second-strand synthesis on the table for the fragments, I do think a recombinase approach might work without too much protein engineering...each fragment has a non-digestable ssXNA sticking out of one end, while the other end is dsDNA; then the recombinases filament on the ssXNA and invade the dsDNA rear end of their matching neighbor fragment, then you have the polymerase a | 19:18 |
| yashgaroth | nd ligase come in, while the leftover third strand on the invaded fragment fucks off and gets degraded | 19:18 |
| kanzure | do all the recombinases work the same way. wikipedia's articles about recombinases are super bad. | 19:20 |
| yashgaroth | sooort of, I'm mostly familiar with phage ones since they work a lot faster than higher lifeforms'; been trying to find a good picture of the mechanism | 19:21 |
| yashgaroth | if you GIS strand invasion there's the basic idea, the leftover ssDNA gets coated with recombinase and then shoved into a dsDNA duplex where the sequence matches | 19:23 |
| yashgaroth | normally you're invading the middle of the dsDNA to do PCR amplification, but it works at the blunt end too | 19:24 |
| kanzure | and what is the sequence match length..? | 19:24 |
| kanzure | or, max match length. or minimum. | 19:25 |
| yashgaroth | it works well with PCR-length primers, so people mostly use 24-30, there should be data somewhere on upper/lower limits | 19:25 |
| yashgaroth | minimum is ~6 since each recombinase protein covers about that many nucleotides, not sure how efficient it is at that length | 19:25 |
| yashgaroth | ah "Oligonucleotides of up to 45 nucleotides have been successfully used as primers [...]and primers could even longer. However, lengthening the oligonucleotide does not necessarily improve the amplification performance and it increases the likelihood of secondary-structures that could lead to primer noise | 19:28 |
| yashgaroth | problem is then you need a specific 5'-3' exonuclease to chew off the leftover third strand since if you just polymerize off the invading strand it'd displace the invaded fragment's ssDNA extension, so it couldn't invade the next fragment in the chain | 19:42 |
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| yashgaroth | my apologies for the quality http://imgur.com/a/Xe967 | 19:54 |
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| kanzure | :) | 19:56 |
| kanzure | yashgaroth: on step 2, is that a smudge or is that an extra line. and on step 3, is the slanty line the non-digestable XNA? | 19:58 |
| yashgaroth | smudge; the slanty line is the displaced third strand from the invaded dsDNA which is shoved off the duplex by the recombinase | 19:59 |
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| yashgaroth | tbf the ssDNA doesn't even need to be XNA now that I think about it, if the exonuclease is specific to one orientation...but I don't trust exonucleases on their specificity | 20:00 |
| yashgaroth | also not sure if the exonuclease will be processive enough to chew off the third strand perfectly, but you'd only have a nick-repair ligase so no ligation between dsDNA blunt ends | 20:01 |
| kanzure | i really dislike all the crazy edge cases that dna manipulation enzymes have :( | 20:02 |
| yashgaroth | also even with XNAs the whole ssDNA doesn't need to be XNA, just the last few bases so the exonuclease can't start chewing on it | 20:03 |
| kanzure | ah that is good. lots of XNA synthesis chemistry would be annoying. | 20:03 |
| yashgaroth | yeah I tried to get mung bean nuclease to do one simple job for like two weeks, then just gave up and got the sequence synthesized | 20:03 |
| yashgaroth | usually people do the last 2-3 as XNAs, phosphorothioates | 20:04 |
| kanzure | oh. what? XNAs are in common use? | 20:05 |
| yashgaroth | but if exonuclease T is specifically 3'-5' as claimed, then maybe it can be avoided | 20:05 |
| yashgaroth | yeah mostly to protect primers from degradation in that case | 20:05 |
| yashgaroth | lots of polymerases have an exonuclease component for proofreading, sometimes it's a separate domain you can remove, so even best case scenarios it's sometimes better to XNA it up | 20:06 |
| yashgaroth | oh I suppose modifying the backbone doesn't count as an XNA *technically* but whatever | 20:07 |
| yashgaroth | anyway forget I said XNAs since we may not need them for this, and I'm reading into the nomenclature for modified nucleic acids and ughhhh | 20:10 |
| yashgaroth | k http://imgur.com/a/ahFmB | 20:28 |
| yashgaroth | aw fuck I misspelled "assembly", well I'm out of paper | 20:32 |
| kanzure | er. now why aren't people doing this already? heh. | 20:34 |
| yashgaroth | I would've had no idea recombinases existed, or their capabilities, if I hadn't been paid to read papers about them for the past year | 20:35 |
| kanzure | what? but you tend to know things, and such. | 20:36 |
| kanzure | and also, wasn't this the whole basis for the 'recombinant dna editing' stuff from the 70s | 20:36 |
| yashgaroth | bro there's like, over a hundred enzymes out there...anyway, people spend their whole lives in biology pigeonholing themselves | 20:36 |
| yashgaroth | nah, the stuff in the 70's was just restriction enzymes | 20:37 |
| kanzure | hm. this is all very troubling. | 20:37 |
| kanzure | i agree with your assessment about biologists tending to pigeonhole themselves, sure. it seems accurate. | 20:39 |
| yashgaroth | and of course perhaps someone tried it and it didn't work, and they preserved their honor by publishing nothing | 20:39 |
| kanzure | and it's not like i have a good index of all the dna editing enzymes either, sooo..... i should fix this. | 20:40 |
| kanzure | i spent a day once and read through all the pdb entries. there were only like 20,000 or something at the time. | 20:41 |
| kanzure | er i mean <200,000 | 20:42 |
| kanzure | http://www.rcsb.org/pdb/statistics/contentGrowthChart.do?content=molType-protein&seqid=100 | 20:42 |
| kanzure | ah i'm thinking of unique proteins. and that page says ~200,000 structures (not unique structures). | 20:42 |
| yashgaroth | sometimes I imagine Gibson as a toddler, playing with enzymes instead of toys, and he mashes t5 exonuclease into a polymerase and money falls down from the ceiling | 20:42 |
| yashgaroth | yeah there's a ton of repetitive pdb entries | 20:43 |
| kanzure | i always felt bad about not paying enough attention to zinc finger nucleases | 20:52 |
| kanzure | .wik zinc finger nucleases | 20:53 |
| yoleaux | "Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes." -- https://en.wikipedia.org/wiki/Zinc_finger_nucleases | 20:53 |
| kanzure | .wik TALEN | 20:53 |
| yoleaux | "Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands)." -- https://en.wikipedia.org/wiki/TALEN | 20:53 |
| kanzure | .wik CRISPR | 20:53 |
| yoleaux | "Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacteriophage virus or plasmid." -- https://en.wikipedia.org/wiki/CRISPR | 20:53 |
| kanzure | .wik recombinase | 20:54 |
| yoleaux | "Recombinases are genetic recombination enzymes. DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression." -- https://en.wikipedia.org/wiki/Recombinase | 20:54 |
| kanzure | man these are bad summaries. gosh. | 20:54 |
| yashgaroth | much as I love to hate on CRISPR hype, using a nucleid acid guide is much easier than making a new protein every time | 20:54 |
| kanzure | pffft you're just saying that because you don't want to live your life behind a chromatography column and protein expression cultures | 20:55 |
| yashgaroth | better than the 80% of biologists toiling in the ELISA mines, but yes very true | 20:56 |
| kanzure | fenn: the htmlizer stuff is not active due to server reboot | 20:59 |
| kanzure | yashgaroth: someone wants to sequence that alaska frog that does slow metabolism and freeze/thaw during winter, do you have sequencer access that i could pay for? | 21:01 |
| yashgaroth | nope, I just use sanger sequencing places for confirming inserts... that's gonna be someone with a bank of illumina machines | 21:05 |
| kanzure | okay i will bug various people. i figured i should know at least one person that wouldn't mind receiving a entire frog in the mail. | 21:06 |
| yashgaroth | I think whole genome sequencing for animals still gets published on its own; exome sequencing maybe would be more reasonably priced | 21:07 |
| kanzure | hm? 40-80x coverage should be like <$5k at this point. | 21:07 |
| kanzure | ..right? | 21:07 |
| yashgaroth | for organisms previously sequenced, maybe | 21:08 |
| kanzure | what kinda coverage for new shit? | 21:08 |
| yashgaroth | no idea, for new genomes it'd cost six figures probably to get decent coverage | 21:09 |
| kanzure | where are all those magic dna sequencing nanopores? gah. | 21:10 |
| kanzure | i was promised magic | 21:10 |
| yashgaroth | here's some recent corn-seq "Wales and his colleagues sequenced the ancient maize sample, dubbed Tehuacan162, to an average 1.7x depth of coverage, or to 6x coverage for the regions accessible via short reads | 21:11 |
| kanzure | corn? i don't hvae time for corn. | 21:12 |
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| yashgaroth | could start with exome sequencing, and I guess Xenopus frog was sequenced forever ago so that'd be a decent template | 21:13 |
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| fenn | i think the output of fileschanged is different now | 21:18 |
| fenn | test | 21:20 |
| kanzure | also the 15th probably has to be regenerated | 21:21 |
| fenn | ok | 21:25 |
| fenn | fixed | 21:25 |
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