--- Log opened Sat Nov 16 00:00:51 2024
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06:58 < cslr> are here ppl interested in hacking brains?
06:58 < cslr> I have developed software to alter brainwaves using audiovisual stimulation (requires EEG-device and *FAST* CPU)
06:58 < cslr> https://www.blackclinic.net
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07:00 < kanzure> cslr: sorry, only interested in direct brain stimulation, transcranial ultrasound, focused or phased array radiation beams, implanted electrodes, surgical, etc.
07:01 < cslr> audiovisual stimulation has no side-effects when it happens through senses!
07:03 < cslr> many treatments approaches are bad because they believe in treatments' side-effects and don't do much brain measurements
07:04 < cslr> I can often get 10% change in EEG-metrics with audiovisual stimulation and max results I have got was 37%
07:08 < cslr> only problem is that audiovisual stimulation effects disappear quickly when video-stimulation stops (TODO: should try to change long-term memories)
07:12 < cslr> thought MIT researcher have gotten results that isochronic stimulation at 40 Hz can help against alzheimer's..
07:20 < cslr> for example, you can increase intelligence using audiovisual stimulus, by telling results (LLM) from game theory and philosophy to patient (which may then change his thinking if he wants to behave optimally)..
07:20 < cslr> or you can try to brainwash patient using video..
07:22 < cslr> but LLMs online stop creating brainwashing stories and I haven't have money to buy servers to run your own LLM..
07:26 < cslr> audiovisual stimulation is not strongly regulated so there are less problems or no need for expensive hardware
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09:28 < Guest69> Hello, could someone explain where i can find more information about synthetic biology DBTL cycle? - Thanks!
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10:08 < okelano> good evening
10:09 < okelano> could anybody suggest new literature on molecular computing and biocomputing some tools for invivo genetic circuit modification using SEVA 4.0 architecture for theranostic plasmid construction? - thank you.
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16:41 < hprmbridge> joedna_44104> If only microcenter sold biocompatible resins for SLA...
17:14 < hprmbridge> kanzure> where did you new users find us from?
17:22 < hprmbridge> joedna_44104> x.com?  It's posted on your profile.
17:22 < hprmbridge> kanzure> ok just curiosu
17:32 < hprmbridge> joedna_44104> Saw you were talking about enzymatic synthesis and I'm sort of working on that as well, so I figured there might be something interesting here.  It's been a while since I took biology and biochem and I'm trying to catch up on what I've missed.
17:35 < hprmbridge> kanzure> ah, yeah we have a novel method of enzymatic DNA synthesis
17:35 < hprmbridge> kanzure> targeting 100x faster synthesis + 100x longer
17:36 < jrayhawk> is there a public mirror of your twitter postings somewhere?
17:39 < hprmbridge> kanzure> not to my knowledge
17:48 < hprmbridge> joedna_44104> I thought so too.  Even shared the idea with a VC friend of mine and a couple of months later, a new start up for synthesis magically appeared :(. But, some of the algorithms/protocols I was working on were discovered years before.  A lot of the patents out there are easily invalidated by prior art or expired patents.
17:51 < hprmbridge> joedna_44104> The problem that I'm running up against is that enzymes are very expensive.  Also, if you want to get the pre-cloned salt precipitatable his-tagged enzyme plasmids, it appears you need an institutional address either to buy them from addgene or get them from bionet.
17:51 < hprmbridge> joedna_44104> https://stanford.freegenes.org/
17:52 < hprmbridge> joedna_44104> No room for garage biohackers :). I even got turned down for an executive PhD because I mentioned biohacking...
17:58 < hprmbridge> kanzure> what did you want to study or do in particular?
18:02 < hprmbridge> joedna_44104> Synthetic biology/microbiology.   I'm mostly interested in restriction enzymes.  I figured theres a few methods out there to synthesize dna oligos based on them and I wanted to show that there's a group that's closed on standard cloning techniques that will allow synthesis and sequencing using only enzymes.
18:02 < hprmbridge> joedna_44104> further, it should be able to be bootstrapped.
18:03 < hprmbridge> kanzure> I agree that restriction enzymes are largely overlooked these days, but I'm curious why you wanted to focus on them
18:06 < hprmbridge> joedna_44104> because you can make them.  Take Camena bioscience for example.  They have a method to iteratively add n base pairs to a growing strand using a specially crafted dna sequence that's terminated in a hairpin on one side and a IIs restriction site on the other that leaves behind 3-4 bases per blunt ligation and restriction digest.  So you take 64-256 unique dna strands and blunt ligate the one that
18:06 < hprmbridge> joedna_44104> matches the next 3/4 pairs and then digest with Golden Gate style enzymes.  BsaI/SapI.
18:08 < hprmbridge> joedna_44104> If you're smart, you can make those strands with DH5A and the enzymes with TOP10/BL21 and not have to buy special TdT or terminal base pairs.  No clue how Camena Bioscience is going to get around that...
18:12 < hprmbridge> joedna_44104> And then you can use regular old restriction enzymes to sequence dna by matching the terminal residues on an unknown set of dna with the balance of the matching restriction enzyme's binding sequence and just pick a set--like 16--for all 2 base pair iterations and blunt ligate a strand with that balance to the unknown dna strand, determining if it cuts as an indication of either or both ends having
18:12 < hprmbridge> joedna_44104> that terminal residue.  EcoRI would tell you that you have GA at the end or the beginning of the strand(complement) by blunt ligating ATTC(N) and watching to see if you get digestions.  That's a mathematical closure.
18:16 < hprmbridge> joedna_44104> there are means of performing incremental digestion, if you don't have any other information about the strands you're sequencing--i.e. some other Restriction Enzyme cuts at a known site, so you know at (cut site -(length of end digested strand -1)) there is a pair of bases that match a given IIRE binding site.
18:18 < hprmbridge> yashgaroth> using restriction enzymes for sequencing sounds horrible
18:18 < hprmbridge> joedna_44104> Yeah, you need Agar and TAE along with an indicator, but it's mostly closed on enzymes and dna.
18:20 < hprmbridge> yashgaroth> like a thousand times slower than nanopore or illumina or even pacbio. And REs aren't infinite, the known ones only cut a subset of DNA sequences
18:20 < hprmbridge> joedna_44104> Bud, I spent 4 grand on an Oxford Nanopore for my birthday.  If I could get the freegenes set and enough money to synthesize the strands I'd need to do the synthesizing, it would be way cheaper.  900 bucks for a flow cell...
18:21 < hprmbridge> joedna_44104> I have a full set of 16 that match all NN combinations of base pairs.  Whether they are cloned into a plasmid with a his tag to make extracting them easy or not is another story.
18:23 < hprmbridge> yashgaroth> yeah making restriction enzymes isn't hard so there is that
18:25 < hprmbridge> joedna_44104> NNNNNNN(ATTC).... if it cuts, the last NNs are GA.  Hindiii.  GTCC(N) on the blunt ligating strand.  IF NNNNNNN(GTCC) cuts, the terminal residues are AA.  I just opened the enzyme page on snapgene and went right down the line.  AA, AT, AC, AG, etc.
18:26 < hprmbridge> joedna_44104> The only reason to have additional residues to the right of (GTCC) for example is to make it easier to clone/pcr copy and resolve with Agar instead of PAGE.
18:27 < hprmbridge> yashgaroth> camena's stuff is more about assembly, so chemically synthesizing short oligos that can assembled into gene-length products. Some stuff about reversible terminators, I haven't bothered to parse their patents. You won't be making or extracting those from DH5a. Actually wait I don't know how NEB makes their restriction enzymes since the vast majority of them will chew up an E. coli genome. Maybe
18:27 < hprmbridge> yashgaroth> they do periplasmic or something, which makes it more annoying
18:28 < hprmbridge> joedna_44104> I'm assuming that stanford's plasmids express without nuking E. Coli's genome.
18:28 < hprmbridge> yashgaroth> sequencing with agar and restriction digests will kill any throughput you have. IIS enzymes might be useful in assembly with camena's convoluted system but that's about it
18:32 < hprmbridge> joedna_44104> Camena attaches to a magnetic bead on the growing strand.  The donor strand with the terminal IIS site is hairpin terminated to avoid complement ligating.  That hairpin can be made via T7 (enzyme?) so you're cloning in the 16-256 strands of dna--one per customer--into a plasmid and copying it, or just making the matching primers and PCRing it, but with the cloning, the bacteria make your
18:32 < hprmbridge> joedna_44104> dNTPs...The restriction sequencing idea is just to show that you can both read and write with just enzymes and a means to measure length.
18:34 < hprmbridge> yashgaroth> that's not writing with enzymes, it's assembly
18:34 < hprmbridge> joedna_44104> Josie is getting herself in to some things that may cause unwanted attention.  Unwanted to the point that Sanger reagents will be regulated and watched.  hell, you'll not be able to order agarose off of Amazon soon.
18:35 < hprmbridge> yashgaroth> I'm assuming RE expression in e coli just nukes the cells, but you can still recover protein from that. Albeit not much
18:35 < hprmbridge> yashgaroth> lmao no
18:35 < hprmbridge> joedna_44104> You're adding 3-4 bases per assembly step instead of a whole gene or set of oligos.
18:36 < hprmbridge> joedna_44104> and that's reasonable to the point of making arbitrary sequences of DNA from a pool of 64-256.
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18:38 < hprmbridge> yashgaroth> jo is making wacky animals, or rather will spend years trying to get a mouse to grow something that looks like a horn. No one has used sanger for the last 20 years so that wouldn't really be a concern. I wouldn't trust amazon agarose for electrophoresis anyway, but that's just me
18:39 < hprmbridge> joedna_44104> http://freegenes.github.io/genes/BBF10K_003281.html and http://freegenes.github.io/genes/BBF10K_003282.html give you EcoRI and the methyltransferase.
18:49 < hprmbridge> joedna_44104> Right, but if they lock down everything, Sanger would be looking pretty great.  I mean, Mixael Laufer's synthesizing schedule I drugs with his microlab.  How long till everything more complicated than ethanol and methanol is going to be restricted?  I grew up in the era where my friend and I could make nitroglycerine in the back yard.  In Texas, if you're caught with an erlenmeyer and no licence,
18:49 < hprmbridge> joedna_44104> you're in trouble.  Ever read the legal section of sciencemadness?  Just because you may have access to what you want in your day job, doesn't mean that you might not want a way to do your own thing without PI or accounting scrutinizing your receipts.
19:44 < hprmbridge> joedna_44104> I get the feeling we're approaching this topic from different points of view.  From your write up on that hack 'inject plasmids bro' company, I guess you've got quite a bit more biology background than I do.    I stopped in undergrad and mostly focused on physics then shifted to computer science.
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20:36 < okelano> Good morning
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--- Log closed Sun Nov 17 00:00:52 2024