| --- Day changed Thu Dec 25 2014 | ||
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| maaku | kanzure: libbitcoin is a C library... | 00:59 |
|---|---|---|
| DumpsterD1ver | 01:36 | |
| poppingtonic | http://slur.io/ | 01:59 |
| poppingtonic | .title | 01:59 |
| yoleaux | Introducing Slur | 01:59 |
| poppingtonic | "Slur is written in C and operates over the Tor network with bitcoin transactions through libbitcoin. Both buyers and sellers are fully anonymous and there are no restrictions on the data that is auctioned. There is no charge to buy or sell on the Slur marketplace except in the case of a dispute, where a token sum is paid to volunteers." | 02:00 |
| poppingtonic | http://www.cell.com/cell/fulltext/S0092-8674(14)01566-9 | 02:05 |
| poppingtonic | .title | 02:05 |
| yoleaux | poppingtonic: Sorry, that doesn't appear to be an HTML page. | 02:06 |
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| kanzure | .title | 07:42 |
| yoleaux | kanzure: Sorry, that doesn't appear to be an HTML page. | 07:42 |
| kanzure | still? | 07:42 |
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| kanzure | quack | 07:51 |
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| kanzure | tRNA synthetases look like a good target for a wash-based protocol, might be better than a tdt-based dna synthesis wash-based protocol | 08:36 |
| kanzure | insert 1 of 20 tRNA synthetase, insert amino acid, heat/wait, wash, repeat. er, will require the tRNA part to be broken/busted/mutated | 08:37 |
| kanzure | (or to have a tRNA library, and break the mRNA/tRNA recognizer components elsewhere) | 08:37 |
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| kanzure | ParahSailin: is there an in vitro protein synthesis that does not require dna, mrna, or trna? | 10:48 |
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| kanzure | you could do in vitro selection of ribozymes to get specific peptidyl transferases to join amino acids by a peptide bond, and maybe some sort of cap, so that you can do a wash step to remove the ribozymes and the amino acids and repeat the cycle | 10:58 |
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| kanzure | also, those "de novo" peptide synthesizers- do they have peptide length limits as bad as the oligonucleotide synthesizers? | 11:04 |
| yashgaroth | much worse | 11:05 |
| kanzure | "SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility.[citation needed] Synthetic difficulty also is sequence dependent; typically amyloid peptides and proteins are difficult to make. Longer lengths can be accessed by using native chemical ligation to couple two peptides together with quantitative yields." | 11:05 |
| kanzure | yashgaroth: what about in vitro protein synthesis stuff? what's the limit | 11:05 |
| yashgaroth | like, cell-free but with rna/ribosomes? afaik you can go as large as in vivo stuff, just the quantities are tiny | 11:07 |
| kanzure | excellent | 11:07 |
| kanzure | screw dna synthesis, let's do that one | 11:07 |
| yashgaroth | what do you need nanograms of protein for | 11:07 |
| kanzure | in vitro protein synthesis needs to be achieved without dna/rna | 11:07 |
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| kanzure | so you can manufacture polymerases | 11:08 |
| kanzure | if you can get rid of the rna/dna requirements then you can just do wash cycles and dump in amino acids + the right transferases or w/e | 11:08 |
| yashgaroth | oh man if you thought it was difficult with 4 components, 20 is gonna be something else | 11:08 |
| kanzure | the difficulty with dna synthesis was not the number of components | 11:09 |
| kanzure | did you ever take a look at cathal's proposal to use telomerases? https://groups.google.com/d/msg/enzymaticsynthesis/6GZT8zFNOfo/npfYUhf1HJ8J | 11:11 |
| kanzure | i'm also curious to hear your thoughts about this oligonucleotide synthesis method using terminal deoxynucleotidyl transferase http://2014.igem.org/Team:Cooper_Union/TdT_project | 11:12 |
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| yashgaroth | perhaps I need a closer look at the telomerase one, but if you have 3+ of the same base in a row, wouldn't you get runaway addition? will check out the igem one now | 11:14 |
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| kanzure | i think ecoli dna polymerase 1 is like >1500 bp | 11:23 |
| kanzure | which is not achievable using traditional single-strand-style dna or oligo synthesis | 11:23 |
| kanzure | but should be achievable with in vitro protein synthesis | 11:23 |
| yashgaroth | hmm well the t/2 of the heat-labile protecting group is five minutes which means a long-ass time if you want ~99% deprotection, and the handwaving of "oh we'll just find or engineer a TdT that can survive 95C for hours" makes me annoyed | 11:24 |
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| kanzure | if you believe that enzymatic synthesis is going to always be the most productive way to do dna synthesis, then it makes sense to go after enzyme synthesis first | 11:30 |
| kanzure | plus, in vitro protein synthesis (sans dna and sans rna) gets you a bunch of capability anyway | 11:31 |
| yashgaroth | you mean the coding sequence of pol 1 being >1500? you could make it with cell-free, but not chemical construction | 11:31 |
| kanzure | right | 11:31 |
| kanzure | i'm talking about making up some cell-free method that doesn't need dna or rna | 11:32 |
| kanzure | really the ribosome just needs a transferase to give it something, right? | 11:32 |
| yashgaroth | very theoretically | 11:33 |
| kanzure | there is some sort of matching/coordination that happens, which would have to be disabled | 11:33 |
| yashgaroth | to say the least, yes | 11:35 |
| yashgaroth | there's hundreds of proteins which'll add basepairs that you can sample/modify from nature, but only one that adds amino acids; not to discourage | 11:41 |
| kanzure | you could maybe make tRNA synthetases that have "locked" tRNA | 11:44 |
| kanzure | weird, it binds an amino acid first | 11:45 |
| kanzure | "The synthetase first binds ATP and the corresponding amino acid (or its precursor) to form an aminoacyl-adenylate, releasing inorganic pyrophosphate (PPi). The adenylate-aaRS complex then binds the appropriate tRNA molecule, and the amino acid is transferred from the aa-AMP to either the 2'- or the 3'-OH of the last tRNA nucleotide (A76) at the 3'-end." | 11:45 |
| yashgaroth | locked at which stage? | 11:46 |
| kanzure | so, actually, you could separately prepare this synthetase enzyme coupled to tRNA and coupled to an amino acid | 11:46 |
| kanzure | (since that's how it seems to happen in the cell anyway) | 11:46 |
| kanzure | "The product of this reaction is an aminoacyl-tRNA. This aminoacyl-tRNA is carried to the ribosome by EF-Tu, where mRNA codons are matched through complementary base pairing to specific tRNA anticodons. Aminoacyl-tRNA synthetases that mispair tRNAs with the wrong amino acids can produce mischarged aminoacyl-tRNAs, which can result in inappropriate amino acids at the respective position in protein. This "mistranslation"[3] of the genetic ... | 11:47 |
| kanzure | ... code naturally occurs at low levels in most organisms, but certain cellular environments cause an increase in permissive mRNA decoding, sometimes to the benefit of the cell." | 11:47 |
| kanzure | "The ribosome has three sites for tRNA to bind. They are the aminoacyl site (abbreviated A), the peptidyl site (abbreviated P) and the exit site (abbreviated E). With respect to the mRNA, the three sites are oriented 5’ to 3’ E-P-A, because ribosomes move toward the 3' end of mRNA. The A site binds the incoming tRNA with the complementary codon on the mRNA. The P site holds the tRNA with the growing polypeptide chain. The E site ... | 11:48 |
| kanzure | ... holds the tRNA without its amino acid. When an aminoacyl-tRNA initially binds to its corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the tRNA in the A site and the amino acid of the charged tRNA in the P site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA in the P site, now without an amino acid, to the E site; ... | 11:48 |
| kanzure | ... the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P site. The tRNA in the E site leaves and another aminoacyl-tRNA enters the A site to repeat the process.[4]" | 11:48 |
| kanzure | hrmm. | 11:48 |
| kanzure | [3] is http://www.ncbi.nlm.nih.gov/pubmed/25220850 | 11:49 |
| yashgaroth | yeah it's a clusterfuck inside ribosomes | 11:50 |
| kanzure | so you could probably get a ribosome that does mistranslation by having it try to synthetize a ribosome poison. then you select the dna/rna-tagged ribosomes that are not denatured. but this is a large project itself... | 11:51 |
| kanzure | *synthesize | 11:51 |
| kanzure | huh, some antibiotics may cause mistranslation | 11:53 |
| yashgaroth | many definitely do | 11:54 |
| kanzure | "In addition, streptomycin was found to simultaneously stimulate tRNA selection errors and processivity of translation for a mutant that is partially dependent on streptomycin." | 11:54 |
| kanzure | from "Ribosome Mutants with Altered Accuracy Translate with Reduced Processivity" http://www.sciencedirect.com/science/article/pii/S0022283685702422 | 11:54 |
| kanzure | .title http://www.nature.com/nsmb/journal/v14/n1/abs/nsmb1183.html | 11:59 |
| yoleaux | Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome : Abstract : Nature Structural & Molecular Biology | 11:59 |
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| kanzure | here are some thoughts about error-prone mutant ribosomes http://www.sciencedirect.com/science/article/pii/S1097276510004508 | 12:01 |
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| kanzure | .tite http://pubs.acs.org/doi/abs/10.1021/cb5006596 | 12:11 |
| kanzure | "Mycobacterial mistranslation is necessary and sufficient for rifampicin phenotypic resistance" | 12:15 |
| kanzure | .title http://pubs.acs.org/doi/abs/10.1021/cb5006596 | 12:15 |
| yoleaux | An Error Occurred Setting Your User Cookie | 12:15 |
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| kanzure | "How about this: degenerate mRNA nucleotides and orthologous tRNAs, so an arbitrary "next triplet" can be loaded with a chosen amino? To avoid repeats have two incompatible triplets staggered and two sets of 21 aminos per triplet. Feed charged tRNA stepwise, wash between. No modified ribosome required, minimal modified tRNAs." | 14:27 |
| kanzure | having trouble figuring out why "two sets of 21 aminos per triplets".. so 21 different triplets, and then 21 aminos per triplet? | 14:29 |
| kanzure | so 21*21? | 14:29 |
| kanzure | what am i multiplying? | 14:29 |
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| kanzure | ".... nonribosomal peptide synthetases (NRPS) (81, 111). In contrast to ribosomal peptide synthesis, non-ribosomally assembled peptides contain not only the common 20 amino acids (aa) but hundreds of different building blocks. Moreover, these secondary metabolite peptides contain unique structural features, such as D-amino acids, N-terminally attached fatty acid chains, N- and C-methylated residues, N-formylated residues, heterocyclic ... | 16:54 |
| kanzure | ... elements, and glycosylated amino acids, as well as phosphorylated residues (111)." | 16:54 |
| kanzure | "Molecular mechanisms underlying nonribosomal peptide synthesis: approaches to new antibiotics" | 16:57 |
| kanzure | .title http://mmbr.asm.org/content/70/1/121.full#ref-111 | 16:59 |
| yoleaux | Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides | 16:59 |
| kanzure | .title http://www.sciencedirect.com/science/article/pii/S1074552111002742 | 17:11 |
| yoleaux | kanzure: Sorry, that doesn't appear to be an HTML page. | 17:11 |
| kanzure | "The error committed by aa-tRNAsynth never exceeds once in 104 | 17:14 |
| kanzure | enzymatic cycles." | 17:14 |
| kanzure | er, 10^4 | 17:14 |
| kanzure | according to http://arxiv.org/pdf/1212.6827.pdf | 17:14 |
| kanzure | "NRPS-mediated polypeptide synthesis does not use mRNA as a template. Instead, the identity and the order of the protein domains of the synthetase serve as the template [161, 174]. In each module of a NRPS one (or two) of the leading domains serve as “gate-keeper” and specifies the identity of the monomer to be selected. Thus, in contrast to polynucleotide templates discussed in the preceeding sections, proteins serve as the ... | 17:17 |
| kanzure | ... templates for NRPS-mediated polymerization. " | 17:17 |
| kanzure | "Nonribosomal peptides are synthesized by one or more specialized nonribosomal peptide-synthetase (NRPS) enzymes. The NRPS genes for a certain peptide are usually organized in one operon in bacteria and in gene clusters in eukaryotes. However the first fungal NRP to be found was ciclosporin. It is synthesized by a single 1.6MDa NRPS.[4] The enzymes are organized in modules that are responsible for the introduction of one additional amino ... | 17:23 |
| kanzure | ... acid. Each module consists of several domains with defined functions, separated by short spacer regions of about 15 amino acids." | 17:23 |
| kanzure | from http://en.wikipedia.org/wiki/Nonribosomal_peptide | 17:23 |
| kanzure | well that's interesting, but basically those are only going to be useful if you have dna/rna synthesis already | 17:26 |
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| kanzure | .title https://news.ycombinator.com/item?id=8796270 | 17:45 |
| yoleaux | The Fermi Paradox | Hacker News | 17:45 |
| kanzure | (maaku made some good comments there) | 17:45 |
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| kanzure | .title http://www.pnas.org/content/72/6/2193.short | 17:58 |
| yoleaux | Enzymes as reagents in peptide synthesis: enzymatic removal of amine protecting groups | 17:58 |
| kanzure | ^v. cool | 17:58 |
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| kanzure | also cool: Enzymatic protecting group techniques http://www.kois.sk/bioorg/bioorganicka_chemia/BIOORG2/3367-enz%20protect%20groups.pdf | 18:00 |
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| kragen | it looks like in addition to the traditional electrical discharge machining, for metal cutting by vaporizing it with arcs in distilled water, and the electrochemical machining fenn told me about the other day | 18:32 |
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| kragen | there's something called electrochemical discharge maching, or electrochemical spark machining, which can be used for machining ceramics and glass | 18:32 |
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| kragen | this paper I'm reading talks about using a 500-micron-diameter platinum-wire cathode in a 14%-20% sodium hydroxide solution with a non-consumable big honking graphite anode over on the other side of the cell to cut channels in glass | 18:38 |
| kragen | I guess you stick the cathode in the solution and the 40-50 volt power supply creates sparks around the cathode, which somehow make the sodium hydroxide solution etch the glass | 18:39 |
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| streety | do you have a link kragen? | 18:41 |
| kragen | I'm looking at http://cdn.intechweb.org/pdfs/27088.pdf right now but that may not be the best thing to look at | 18:43 |
| kragen | so I guess they figure the cathode forms hydrogen bubbles through electrolysis, and then you get sparks across those bubbles | 18:44 |
| kragen | how this affects the glass is still a mystery to me | 18:44 |
| kragen | the paper suggests "electron bombardment" | 18:45 |
| kragen | like apparently the avalanche discharge through the hydrogen bubble acts as a particle accelerator? and then what, you get negatively ionized glass? | 18:47 |
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| kragen | you get hot glass apparently | 18:49 |
| kragen | maybe hot glass is more vulnerable to alkaline erosion | 18:49 |
| ParahSailin | the shady stem cell clinics in south america and thailand, are they using ipsc's or just regular stem cells separated from fat | 18:50 |
| kragen | do you want me to go ask? are there any here in Buenos Aires? | 18:50 |
| kanzure | i haven't seen any evidence that they are using induced pluripotence | 18:50 |
| kanzure | and i think they existed prior to when we figured out induced pluripotence | 18:51 |
| kragen | this other ECDM paper uses a 2mm copper cathode, 155 volts, 5% hydrochloric acid, and 600-micron separation from the workpiece, which was also copper | 18:54 |
| kragen | it says the workpiece surface heated up to 865° C | 18:54 |
| ParahSailin | i dont think there are any in ar, they seem to pick "closer" places like panama or tijuana | 18:56 |
| kragen | that's weird, usually advanced medical stuff happens here before it happens in panama | 18:56 |
| ParahSailin | well panama is a shorter plane ride for most people | 18:56 |
| ParahSailin | pluripotency was figured out in 2006, i would have hoped some shady clinic would have started offering it by now | 18:57 |
| yashgaroth | step right up and get your very own teratoma | 18:58 |
| kanzure | yesterday on the highway i was stuck behind this guy https://www.youtube.com/watch?v=Y-sziu1tWV0&t=54s | 19:06 |
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| kanzure | this could be a good shoe ad https://fbcdn-sphotos-b-a.akamaihd.net/hphotos-ak-xap1/t31.0-8/q83/p843x403/10580814_954540054573165_7697267984601054148_o.jpg | 19:47 |
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| bbrittain | paperbot: http://www.sciencemag.org/content/337/6096/816.short | 20:14 |
| paperbot | http://libgen.org/scimag/get.php?doi=10.1126%2Fscience.1225829 | 20:14 |
| kanzure | .title http://www.sciencemag.org/content/337/6096/816.short | 20:14 |
| yoleaux | A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity | 20:14 |
| bbrittain | it's sorta the original CRISPR/cas paper | 20:15 |
| kanzure | bbrittain: https://groups.google.com/d/msg/enzymaticsynthesis/3YEEv0OULo0/zJZPETWDbMIJ | 20:15 |
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| bbrittain | kanzure: there is an enzymatic synthesis mailinglist? | 20:16 |
| bbrittain | cool | 20:16 |
| kanzure | yes, however the downside is that the ingredients to the enzymatic synthesis mailing list are like 100% hplusroadmap | 20:16 |
| kanzure | so.... just some thoughts we have written down in the past, basically... | 20:16 |
| bbrittain | so, how useful would this be? | 20:25 |
| kanzure | well, i want enzymatic dna synthesis | 20:25 |
| kanzure | because we have proof-of-existence examples of polymerases pooping out entire genomes for <$1 | 20:26 |
| kanzure | since nobody has figured out enzymatic dna synthesis, a suitable temporary alternative would be enzymatic protein synthesis | 20:26 |
| kanzure | solid-phase protein synthesis can only do about 70 amino acids, which while useful has similar problems to dna synthesis (=> only short fragments, sigh) | 20:26 |
| bbrittain | :( | 20:27 |
| kanzure | so i think the protein synthesis method i proposed could have a good chance at printing out huge proteins | 20:27 |
| bbrittain | interesting... | 20:29 |
| bbrittain | hmm | 20:29 |
| yashgaroth | yeah I like that alternating codon idea | 20:29 |
| kanzure | once you have working huge protein synthesis, you could go back to working on dna polymerase or whatever | 20:29 |
| kanzure | and in the mean time you have cool stuff like "print whatever proteins you want" | 20:33 |
| kanzure | i wonder how long a ribosome could be halted before its unable to resume correctly | 20:38 |
| kanzure | adding/removing enzymes at each step might get expensive, heh | 20:42 |
| kanzure | .title https://www.youtube.com/watch?v=TfYf_rPWUdY | 20:54 |
| yoleaux | mRNA Translation (Advanced) - YouTube | 20:54 |
| kanzure | this isn't a completely awful animation. plus, no ribbons. | 20:54 |
| kanzure | .title https://www.youtube.com/watch?v=Jml8CFBWcDs | 20:56 |
| yoleaux | Ribosome in action - YouTube | 20:56 |
| kanzure | this is a better animation | 20:56 |
| kanzure | .title https://www.youtube.com/watch?v=TRIDCQM3d7I&t=4m30s | 21:03 |
| yoleaux | What We Have Learned from Structures of the Ribosome - YouTube | 21:03 |
| kanzure | "tRNA selection is fast (~22 per second) and accurate (>10^3 - 10^6)" | 21:29 |
| kanzure | (at 31m 40s) | 21:29 |
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| jrayhawk_ | biochem porn | 22:40 |
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| nmz787 | I think I am gonna try this for photomask generation, $120 shipped, http://www.aliexpress.com/item/Promotions-DIY-laser-engraving-machine-Mini-laser-engraving-machine-carving-seals-rubber-stamp-mobile-phone/1805621759.html | 23:54 |
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