Transcription Activator-Like Effector Nucleases (TALENs)

okay so in this video what we're going to discuss is the is tailings basically which stands for transcription activator like effects or new crazes okay so let me write that down and basically what tailings are is they are another artificial nuclease that we can use to cut DNA at incredibly specific recognition sites so talen stands for transcription activator like effector nucleases okay so basically these are type of end a restriction enzyme which means that they are going to recognize a specific DNA sequence and basically they're going to cut at that specific DNA sequence okay so let me draw out a DNA sequence and then I'll discuss what they are going to do all right so okay so let's make up some nucleotides so let's say GCT a a GT @ t a a a G C c/g a T C c/g a T how many of you got that 1 2 3 4 of 6 9 12 15 18 I think that's 21 22 so we'll need two more so let's have C and G there right then we'll put in the opposite the convention ones at C c/g a 2 T C a and T 2 k c GG c t a GG c c MF c nope t and a G okay there we go right so basically what what these tailings are made out of is they are made out of transcription activator like effector proteins which are often an often called just tails for short so transcription activator like effectors and basically these are proteins secreted by certain bacteria known as Zam from Onis bacterium okay so Sam fro as an Zam fro Monas species of bacteria secrete these transcription activator like effectors okay and what is the function what are these transcription activator like effectors do well basically they are a protein which binds to DNA and it binds to a specific nucleotide so we are getting very very very nice proteins now because this protein this tail this transcription activator like effector is going to bind not just to a codon I remember in the previous video where we studied zinc finger nucleases these zinc fingers were binding to Hulk who Don's but now these transcription activator like effectors are going to bind a DNA at a specific nucleotide so maybe adenine will have a specific transcription activator like effector which will bind to it by même will have a different transcription activator like effector which will bind to it now this means that we can create very specific nucleuses from this and basically what we do is we have 24 here is that going to work it takes six away from that you get 18 and so then we'll have nine on each side that seems a lot and then you have six yeah it's working fine right okay so basically what you can do is you can use this to build nucleus and this is how you do it you are put in you attach loads of these tails together you attach those of these transcription activator like effectors together to make a horror huge weight protein which has absolutely loads of these tails all together so often you'll join about nine of them together so you put nine of them together now and all the ones that recognize gee I will color in warm color so these are all the same tail which we've we've bonded them all together to make a bigger one but those are all the transcription activator like effectors which bind to gee specifically then you obviously need lots that bind to C so we've got one here that binds to C so this one will be seen and then these ones that bind to T I'll color in green so these are all of transcription activator effector and they're activated like effectors and finally you have the transcription activator like effector which binds to adenine so we have bonded together all of these transcription activator like effectors and now what we have is a great big protein that doesn't just bind to one nucleotide it binds to a whole sequence of nucleotides binds specifically to this sequence of nine you clear tides and now it recognizes that entire sequence and will only bind if that entire sequence of nine nucleotides is there okay now what we do is attach half of an endonuclease enzyme to this so attach half of F ok L now F okay L is again a nucleus which we got from bacteria we got it from a flower bacterium I think so it's it's a we it's an end a new craze that is formed by two parts basically it only becomes active when the two parts dime eyes and then it becomes an active endonuclease okay so you need the two parts to both be there in order for you to have an active enzyme you're right so we attach the other half to a Taven which we're going to attach on well we attach the other half to a bunch of tails which we're going to attach to this other this other this complimentary strand of DNA basically so again what we do is we create a horse we attach a whole sequence of tails together these transcription activator like effectors we touch them all together so they recognize this sequence over here this sequence of nine nucleotides okay so there we go so I'll color those ones in two so the G ones were pink color so let's color them in first so these are all transcription activator like effectors which bind to G and then we had C was orange so all the transcription activator like effectors which bind to see are going to be these ones here okay T was green so all the transcription activator line factors which bind to T will color in green and finally a was yellow so we'll cover in all the transcription activator like effectors which bind to a in yellow so we've now created a whole sequence of a transcription activator like effectors we've bonded them together so that we now have this great big structure this great big protein which recognizes this whole sequence of nine here and we bond that sequence of nine to this other half of this F okay L enzyme and basically what happens is that when we put these two structures that we have engineered into the DNA sample this structure will bind here and this structure will bind here then what will happen is that the two halves of the fok L enzyme will demise they'll form a functional enzyme and that enzyme will then cut these six nucleotides which are in this space in between and it will cut basically in a staggered way so it'll leave sticky ends at it or leave these overhangs basically our four nucleotides but what's brilliant about fok out is that it is not specific in itself if you look at other restriction endonucleases which cut sticky ends like eco r1 eco r1 has a very specific recognition sequence which it has to have in order to cut so it needs g-a-a-t-t-c basically with the complementary one here CTT AAG so basically if we put two halves of eco r1 on here you have to have that specific region in there but basically what's brilliant about fo Keo is that you can have whatever six you can choose whichever combination of nucleotides you like in here basically which is what's very nice okay so that's why we use fo KL so all of the specificity basically is in is in deciding which which combination of transcription activator like effects as you want in these portions of V Taylor okay so this entire thing I all of these nine tails all of these nine tails and the two halves of F okay L that is known as the transcription activator like effector nucleus or the talen for short okay and again just like zinc fingers it is an extremely specific nucleus because as you can see this is going to require a specific combination of of about 18 nucleotides and it's also going to require them to be positioned in specific places so it's going to be very very specific you can make these even more longer if you want so they'll become even more specific and that's the problem with normal restriction endonuclease which you find in nature such as eco r1 that if you put that into the human genome it will just cut all over the place because it will find this combination basically in a lot of places in the human genome whereas you can make these specific enough that they might only cut at one point in the genome so you can target them very very nicely and these are actually preferable to zinc fingers because you can engineer them so well because each one of the tails read that recognizes a single nucleotide whereas when you have zinc fingers recognizing a whole codon it's more difficult to work with you have less flexibility because you know there might not be a zinc finger that binds to 16 for codons so there might be some regions where you can't sign a zinc finger to work so this is much more nice because you know you can build this to recognize whatever sequence you want

Channel: Elliot Nicholson

Views: 64,561

Keywords: Transcription Activator-like Effector Nuclease, transription activator-like effector nucleas, transcription activator-like effector nucleases, TALENs, TALENs biology, TALENs biochemistry, TALENs gene editing, talens gene editing, transcription activator like effector nucleases, transcription activator like effector nuclease, transcription activator-like effector nuclease genome editing, TALENs genome editing, Lecture on talens biology, biology TALENs

Id: qbE-6x_Fgn0

Length: 11min 0sec (660 seconds)

Published: Sun Oct 19 2014