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| kanzure | always with you overlord this and overlord that | 07:34 |
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| adlai | streety: i don't mean to be a shithead, but doesn't politics belong in ##hminusarchaeology ? | 10:11 |
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| kanzure | yes. | 10:13 |
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| kanzure | "Directed evolution relies only on a cycle of introducing diversity into a population followed by the partitioning of that population to isolate the desired function. Theoretically, any population can be systematically optimized towards the desired function by repeating cycles of directed evolution. In practice however, the number of variants in the population can rapidly escalate beyond the sampling capacity of any selection ... | 10:54 |
| kanzure | ... methodology. In addition, as a given functional variant becomes rarer in a population, there is a greater burden on the selection method to isolate them." | 10:55 |
| kanzure | thta's a rather strong claim for directed evolution ("theoretically, any population can be systematically optimized towards the desired function [by directed evolution techniques]" | 10:55 |
| kanzure | ) | 10:55 |
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| adlai | my steelman cortex translates "theoretically, " here as "assuming that phenotypes exist to support the target function, and the genotype landscape to reach them isn't too fissured with valleys of unsurvivability, ..." | 11:08 |
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| kanzure | i am not sure how "the gm3 cultivation device" supposedly prevents biofilm formation https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308500/ | 11:14 |
| kanzure | adlai: i dunno man, that sounds like a pretty big omission to me | 11:14 |
| adlai | they're lucky we're not their peer review! or maybe irc is the real peer review. | 11:14 |
| kanzure | "In one of the earliest examples of eukaryotic continuous evolution, the Evolugator, a proprietary continuous culturing device that reduces biofilm formation and wall growth (Figure 4), was used to evolve thermotolerant filamentous fungi. During long-term continuous culturing of filamentous fungi, wall growth can be problematic as fungal cells rapidly aggregate, leading to non-uniform culture dilution. To circumvent this issue, de ... | 11:15 |
| kanzure | ... Crécy-Lagard and coworkers devised an alternative method for continuous fungi culturing that dramatically ameliorates fungal wall growth [36,37]. Using this platform over the course of four months, the researchers evolved two thermotolerant variants of the entomopathogenic fungus Metarhizium anisopliae to facilitate the widespread adoption of this fungus for pest control [37]. While successful, this method for continuous directed ... | 11:15 |
| kanzure | ... evolution requires specialized equipment that cannot be readily generalized to alternative selection methodologies." | 11:16 |
| kanzure | "... avoid host strain mutagenesis. A similar approach was recently developed by Chang Liu and coworkers using a system that is completely contained in yeast. The authors used an autonomous DNA replication system from Kluveromyces lactis to enable the replication of a gene of interest in the S. cerevisiae cytosol [39]. Due to the strict requirements for the orthogonal K. lactis DNA polymerase initiation, no crosstalk was observed between ... | 11:17 |
| kanzure | ... the K. lactis components and the native host machinery. To make this system more amenable to directed evolution, the authors used a homology-guided approach to create error-prone DNA polymerase variants that would only replicate the orthogonal plasmid. The optimized system was able to induce ~300× higher rates of mutagenesis in the orthogonal plasmid as compared to the background S. cerevisiae mutagenesis rate, with no reduction in ... | 11:17 |
| kanzure | ... the genomic replication fidelity." | 11:17 |
| kanzure | yashgaroth: what is the current status of one pot dna assembly from thousands of fragments with matching overlaps? | 11:36 |
| kanzure | er, where only two molecules in the pot have matching overlaps, i mean. | 11:36 |
| kanzure | i guess i mean golden gate. | 11:37 |
| kanzure | i think it should be possible to mutate and select for an enzyme that is increasingly good at dna ligation based on long 20mer overlap barcodes with very high specificity. you'd start by looking for an enzyme that does random ligation, then you'd move on to selecting for an enzyme that does not quite random ligation, which you can check with dna sequencing. over time you should be able to get to an enzyme that does high-specificty dna ... | 11:45 |
| kanzure | ... end joining. so pcr assembly without primers (or polymerase), i guess. | 11:46 |
| kanzure | oh, then you have barcodes that you have to fix. right... | 11:47 |
| kanzure | ok right, the barcodes could just be the end of the molecules without special random nucleotides. no barcodes needed. however, you would have to make sure that none of your sequences are so short that they would be indistinguishable from each other at the ends. | 11:49 |
| yashgaroth | so you're only assembling two of the fragments, but there's a bunch of random other oligos in the reaction? golden gate's mostly obsolete now, and you're limited with type IIs restriction enzymes | 11:54 |
| yashgaroth | plus there's plenty of enzymes you could add to get it more reliable...for every extra reaction you have happening in the same chamber, you get some exponential or log or w/e increase in potential errors | 11:56 |
| yashgaroth | maybe you get one fragment that's got a single-stranded overhang, homologous to a double-stranded end on another molecule, then use some recombinase enzymes to invade the dsDNA and you fill in the gaps from there | 11:57 |
| yashgaroth | that way you're not relying on sticky end basepairing, since the recombinases actively seek the homologous sequence and hold it on the matching strand until your polymerase or ligase comes along | 11:58 |
| yashgaroth | sorry I've had to do a lot of reading on recombinase machinery for work, people aren't using it for assembly, that I know of, but it's certainly plausible | 12:00 |
| yashgaroth | this is sequence non-specific enzymes, not like Cre or whatever, more RecA | 12:01 |
| yashgaroth | wait are you assembling the whole pot together, just end-to-end with unique matching sequences in between? that's a huge pain | 12:06 |
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| yashgaroth | otherwise you're talking about super-Gibson, no? I do think there's room for proteins that actively seek and match homologous sequences, to actively increase the specificity of that reaction | 12:12 |
| kanzure | err | 12:14 |
| yashgaroth | fill me in before I continue down this tangent | 12:14 |
| kanzure | no no, i just have to switch my brain into biology mode. | 12:14 |
| kanzure | i want one pot with 10000 unique dna sequences (with trillions of molecules or whatever, i don't care). and then i want an enzyme that assembles a long dna molecule by recognizing matching end overlaps. | 12:15 |
| kanzure | you're probably right about recombinases here | 12:16 |
| yashgaroth | so there's phage proteins that form a filament on ssDNA, and then burn ATP to actively scan dsDNA for a matching sequence (maybe they can scan ssDNA, not sure or would need some work) and when they find a match, they invade the dsDNA and hold the ssDNA strand there until e.g. a polymerase comes along and elongates off that primer | 12:17 |
| kanzure | in the imaginary reaction that i am envisioning, most of the short strands would find their matching strands at some point, and the enzyme would do its work. and over time, i would expect the reaction to get less efficient because matching giant dna strands is just physically less likely to happen. | 12:18 |
| yashgaroth | well there is that factor, it's a lot easier with short primers floating around vs. carrying a whole huge DNA molecule, brownian motion etc | 12:19 |
| kanzure | the phage provides a primer? or you mean it's a primer + phage ssDNA | 12:19 |
| yashgaroth | phage protein, so normally you have primers that it attaches to and "scans" with | 12:19 |
| yashgaroth | it's huge in isothermal amplification right now, you have a template but you can amplify exponentially at 37C instead of thermocycling | 12:20 |
| yashgaroth | which allows you to have all sorts of extra enzymes that'd be destroyed by 95C to do other stuff like ligate | 12:20 |
| yashgaroth | so the phage recombinase enzymes replace the heat-melting of the template and amplicon | 12:21 |
| kanzure | neat. | 12:21 |
| yashgaroth | so people use it for that right now, mostly PCR-based detection of pathogens or cancer or w/e, but they could provide actual specificity to sticky-end joining; dunno if anyone's looked into it for that but it's not a terrible idea maybe | 12:22 |
| yashgaroth | otherwise w/ traditional Gibson you have a high chance of a few basepairs sticking randomly and the polymerase don't give a fuck and just fills it in, and you get deletions or mismatches in your final product | 12:24 |
| yashgaroth | so far I only know that they scan a dsDNA target to find homology, and yeah it's a lot faster when it's just primer+enzyme floating around vs. a huge DNA fragment, but it might still be workable | 12:25 |
| yashgaroth | it's like e. coli RecA, in the organism it's not scanning and doing amplification, it's there for DNA repair; but we find a use for things | 12:27 |
| yashgaroth | anyway the generic term is 'recombinase polymerase amplification' | 12:28 |
| kanzure | thanks | 12:35 |
| kanzure | that is helpful | 12:35 |
| yashgaroth | would require some modification for this application, but would be nice to help maintain specificity and reduce off-target ligations | 12:39 |
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| kanzure | hm. | 19:12 |
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| newbie|2 | how many percentual decimals of progress towards singularity would you guys say are performed on a single day? | 20:20 |
| newbie|2 | and, are these decimals less on weekends? | 20:20 |
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| vicarion | i would say that percentage is a complex number. possibly lacking a real component many days | 23:23 |
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| --- Log closed Mon Nov 14 00:00:49 2016 | ||
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