3 ms·

I had a guest lecture from a couple of researchers working on bioproduction that mentioned crispr let them speed up their design process for new e coli strains,

I had a guest lecture from a couple of researchers working on bioproduction that mentioned crispr let them speed up their design process for new e coli strains, but yeah in general it's not too difficult to hack together a pathway with a Gibson (unless you want to do more than 5 parts in the assembly which gets finicky). Or a golden gate if you can get the sequences for the overhangs right. But I will say I've worked in a lab with at least one person who wasnt able to perform a relatively simple gibson somehow, so the principle that the universe will always find a greater fool still applies

dnautics 6y ago
OK, so gibson assembly is not completely foolproof, when I first started working with it I thought the whole thing was a joke because I spent a month trying to get even one clone, and it didn't take. Turned out the site we were trying to (TetR IIRC, it's been 10 years so I'm not sure) clone into had a strange tandem inversion sequence flanking it. If you stick to cloning into MCSes or assembling sequences you've checked/built yourself it should be fine (the liklihood of problematic random tandem inversions is low). a 1:1 cloning is very much doable with terrible hands. You can reasonably do up to 8 pieces in the assembly, but as you get beyond 4 pieces it becomes important to follow the instruction exactly. The craziest thing I did, though, was I cloned a (small) eukaryotic chromosome into an E Coli, by gibsoning against the telomere sequence. At some point the difference becomes using very specially prepared competent cells and being very careful about how you get them in (the mechanism of electorporation is... not obvious), and of course one of the tricky things about gibson is that it's super salty, meaning that you have to be careful when electroporating.

3 ms·

I had a guest lecture from a couple of researchers working on bioproduction that mentioned crispr let them speed up their design process for new e coli strains,

by vikramkr 6y ago

dnautics 6y ago
OK, so gibson assembly is not completely foolproof, when I first started working with it I thought the whole thing was a joke because I spent a month trying to get even one clone, and it didn't take. Turned out the site we were trying to (TetR IIRC, it's been 10 years so I'm not sure) clone into had a strange tandem inversion sequence flanking it. If you stick to cloning into MCSes or assembling sequences you've checked/built yourself it should be fine (the liklihood of problematic random tandem inversions is low). a 1:1 cloning is very much doable with terrible hands. You can reasonably do up to 8 pieces in the assembly, but as you get beyond 4 pieces it becomes important to follow the instruction exactly. The craziest thing I did, though, was I cloned a (small) eukaryotic chromosome into an E Coli, by gibsoning against the telomere sequence. At some point the difference becomes using very specially prepared competent cells and being very careful about how you get them in (the mechanism of electorporation is... not obvious), and of course one of the tricky things about gibson is that it's super salty, meaning that you have to be careful when electroporating.