Engineering yeast to produce spider silk



  • @misslitty that's the plan! just need to get some first. still haven't run the pcr yet though. if that comes back positive then we won't need to.

  • This would be the PCR w shorter primers? Hope it works out!

  • @misslitty thats the one! We're running it today hopefully. will post results when they come back. Also will post a less ugly gel from the extraction

  • Womp womp. Negatory.

    Gonna source some fresher spiders and order more primers and try again

  • ): Can't imagine your frustration. Keep on keeping on, you got this!

  • @misslitty ya it's getting annoying I'll admit. We're planning an easier experiment that's just for fun so we can get at least 1 genetic engineering win to keep us going. I've been keeping busy in the mean time honestly. Been doing some machining and made some cool stuff. Currently building a PCR machine that does gradients and touch down, so that'll be good. Will make doing these reactions easier. Also built a spec, and an ultrasonic homoginizer. Gonna build an electroporator shortly.

    But ya. it'd be nice if this just fuckin worked. The most annoying would be if I have to resort to building it fragment by fragment synthetically. I've been trying to avoid that. The next primer we order will be targeting all the way at the end of the gene so rather than the original plan, we'll be getting a 8000bp fragment if it works. Should deal with the misspriming issue we think we're having. Also no more dimers which we've been getting consistantly. It's huge so it'll be a slow reaction and could be iffy. Once we have it, we can either use it whole, or cut it to size with restriction enzymes. But getting something is the first step. Whether we test that idea on the current spiders or fresh spiders depends on what we end up getting first, fresh spiders, or new primers. Only time will tell. Will keep yall updated

  • God I have so much brain envy, there are no words. I'm assuming it's cheaper to build all of those yourself than to find one someone is trying to get rid of? In any case, that's beyond cool, and having full PCR temp control will be nice!

    Really hoping the new primers solve your issue, it would be a little insane to build it fragment-wise. Looking forward to updates!

  • Yup! a spec would cost 400, we built one for like 40 bucks. An ultrasonic homogenizer would cost over 1k. And a PCR machine with that capability costs hundreds as well. Built the whole set for less than 100 total. Still gonna keep tinkering with all of them though to improve them. The nice part is once they're built it's easy to keep messing with them to get incremental improvements.

    Also on the topic of the spider yeast, chatted with some other genetics people and got some tips and pointers on how to proceed. Gonna rerun the extraction on monday because we think there may actually be intact DNA in the spiders so gonna run a couple tests to find out.

  • edited January 2019
    Thanks for all the footwork you're doing for science, and especially thanks for constantly updating us on your progress. I always look forward to your next post.
  • New video and I discuss the issues with the spiders:

  • New video is up. Not strictly spider silk stuff but its what I've been keeping busy with while I wait for fresh spiders to arrive.

  • This is a very interesting project, any updates @Chironex? I was also curious if you intended on linking individual silk fibers together using transglutaminase or if you have some plan to extract the fibers and spinning them into a thread?

  • Not yet. Waiting on more supplies, building equipment, testing and restesting protocols. Nothing exciting. This is gonna be a slow burner project with lots of fits and starts. The hurdles we have to clear are immense and currently material is hard to come by and slow to arrive. I'm juggling other projects to keep myself busy between mail arrivals

  • Fresh spideys are incoming, along with more supplies.

  • Huh, guess I haven't posted in a while. So, uh, I did the thing

  • I just want to say congrats @chironex! This has been a while in the making! Super happy for you man!!!!

  • Fascinating. I hadn't considered being able to have the silk take in other compounds like that, seems like you've got a really tailorable material on your hands. From the perspective of someone whose main field is aerospace engineering, I'm curious if you could use this to manufacture large directional sheets. Seems like it would be a great alternative to carbon fibre or fibreglass in composite materials, especially as it isn't a galvanic material like carbon. Being able to directly alter the type of fibre (and thus elastic modulus) you use would also have interesting consequences for a whole host of aerostructures problems. Hell, the dream would be to grow an entire wing panel or something, no layup needed.

    Huge accomplishment besides mate, congrats!

  • Awesome! thanks!

  • @chironex said:
    Couple weeks. Last of the supplies should arrive this week, then we'll start on the cloning and plsamid assembly. Should be ready to attempt transfection shortly thereafter. First things first is making sure the species of spider I receive is the same as the gene sequenece I have. If not gotta identify and modify/reorder 1 pair of primers. Should be fine, but we'll see. After that gonna find out if the species of yeast I have matches the integration sequence. Again, should be fine, but I'm planning for the worst case. Other than that the rest should hopefully be smooth sailing. Gonna be doing the first PCR to isolate the g418 resitance probably tuesday and then gibson it into the plasmid later in the week. Just finishing up my new heat block to make that process easier. Once that's done we'll try our first transformation to see if the empty plasmid works. If it does it's spider time

    Haha I'm flattered. Not sure which uni would hire me though XD I like teaching via my channel. All the fun of teaching, without needing to mark exams XD And it lets me regularly publish null results.

    Sorry for late response and for reviving this thread but which channel? Is it a Discord channel? You seem to fascinate me and I would like to talk to you more.

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