The silk game plan (Patreon)

So after much redesign and over a month and a half waiting for reagents to arrive I finally have everything I need to take another crack at the silk project. For a quick summary this is what we've done so far:

Designed a custom silk plasmid that should work in P. pastoris (a species of yeast)

Had it synthesized

Recieved it

Tried half a dozen times to transform it into yeast with no luck thus far

We also took a first pass at trying to cut out the silk gene and stick it into a plasmid that works in ecoli to at least get some silk made while we debug the yeast.  (this alone was weeks of work with little to show for it)

This had been extremely frustrating to say the least and also cost a sphincter tightening amount of money. Many MANY hours and weeks burned in the lab doing all this. But it'll be a cold day in hell before I let this project die so we're pushing forward. I've been putting off making a video about this because I want to show something working rather than the pile of failures and frustration. I have the *theoretically* finished DNA in my freezer but can't seem to cross that final hurdle of making the silk producing yeast. So the latest supply order is designed to rectify that. 

We've now got 3 angles of attack to sort this problem. Route 1, is some changes to the transformation procedure that should hopefully make the stupid yeast take in the DNA. Route 2, is to cut out the silk gene, and insert it into an ecoli plasmid which I know works and I've transformed into ecoli many times already. Just getting the silk DNA itself was a huge challenge and what the first few videos in the series were exploring, so this is actually already significant progress. Now we just need to make something express it and we've got silk. And route 3, is similair to route 2 but ends in a plasmid which should work in a species of yeast which is easier to work with (common bakers yeast). This route will take the longest as it uses the egg white plasmid I designed on a stream a while ago. That plasmid is currently being synthesized so until it arrives I can't pursue this option, but I specifically designed the egg plasmid to make it a possibility to leave my options opened.

So this is my plan for the next week or two (not including work I'm doing on other projects for other videos):

-start turbo (ecoli strain) overnight culture
-start pichia overnight culture
-order bridge spacers (little bits of DNA that act like the in between lego bricks to connect the silk DNA to the bit of DNA we want to insert it into)
-make amp lactose plates
-heat shock and electroporate  cometgfp (testing which method works better, while getting more DNA ready so I have stock to use in later steps)
-digest silk plasmid for ecoli version
-digest silk for pichia transformation (turns out, you need linear DNA for the transformation to work. Hopefully that solves the issue)
-ligate insert (connecting the bridge segments and silk to prepare it to be added to the new host plasmid)
-heat shock pichia (....spider yeast success please?)
-overnight cometgfp (prep for ecoli version)
-extraction cometgfp (more prep)
-digest cometgfp (beginning the process of making the ecoli version. cometgfp is the plasmid the silk is going into. this step cuts it open to accept the insert prepared above)
-ligate silk comet (add silk to backbone. but this step breaks the GFP present in the plasmid. will be fixed later, see below)
-fresh turbo overnight
-transform new silk plasmid into turbo (plasmid is made, get it into ecoli to make copies AND silk!)
-grow overnight silk ecoli (preparing a large stock so we don't lose it)
-extract silk plasmid (extract DNA to prepare the gfp fix I mentioned above)
-digest silk plasmid (cut the plasmid open to insert the fix)
-digest cometgfp (the initial insert process cut out a chunk of the GFP gene present in the plasmid. Here we cut it out again, but save it separately this time so we can put it back into the new plasmid and repair the GFP)
-ligate to repair gfp (create a plasmid that has both silk AND a functional GFP stuck to the end of the silk as a reporter)
-fresh turbo overnight
-transform silk gfp plasmid into turbo (make fluorescent silk producing ecoli)

It's a gonna be a lot of work but it'll be great to finally move forward. Things could fail in about a thousands ways at any of these steps so I doubt it'll be smooth sailing, but fingers crossed, if I can make it to the end of the list, we'll have silk in probably 2 different flavors, ecoli and yeast. The ecoli version is going to be tagged with GFP so if we see glowing colonies, we know that silk is being produced successfully.

That's all for now, just wanted to give ya'll an update. I'm also working on 3 other projects at the same time and will have to start editing one of them into a video next week, so it's gonna be go go go for the next little while.