Genome Manipulation with SacB

Genome Manipulation with SacB

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00:00
all right welcome this is going to be a little recording just to show you how to use some fairly common microbial genetics techniques to manipulate a microbial genome in your way of choosing you can use these techniques to make a clean deletion of an orb within a genome or any region within a genome you can use them to do allelic replacement you can even do them to put new genes into a genome and select for putting new genes into a genome in pretty much any place on the chromosome you would like to do so first things first i'm going to introduce you to the region of the genome that i'm going to be manipulating
00:32
as part of this little module here um i'm going to work with yfg which is your favorite gene the first thing we have to do for actually manipulating the genome is to identify regions upstream and downstream of this yfg region that we actually want to manipulate i've got the upstream region here labeled a i've got the downstream region here labeled b a is in blue b is in green uh it's important to identify these bracketing regions to the region you actually want to manipulate
01:03
because those are going to be the ones we use to actually homologously recombine uh the plasmid into the genome and take advantage of the ability of homologous recombination to kind of move around regions of dna where we'd like them to be all right how long do or how large do these regions have to be the larger the better long story short the larger the better it's homologous recombination we're dealing with so the larger the region that we can do on either end of the region we want to manipulate uh the easier it's going to be to actually select for integration of
01:33
plasmids into the genome and to select for plasmids to flip out of the genome and hopefully the way we we want right typically when i do this when i build them within the genomes i'm using about 500 base pairs on each side of the region i want to manipulate i've gotten away with about 100 base pairs on each side you can even go a little bit shorter than that it just makes your life a little bit more difficult when you're trying to manipulate the genome you typically go 500 base pairs but again the larger the better you know thousands of base pairs would
02:04
be even better than 500 base pairs here all right so we've got region a upstream region b downstream we'll say it's 500 base pairs each of those um and we'll kind of go from there so what do we actually want to do to manipulate this genome um so i'm kind of fond of making clean deletions within genomes to actually knock out my worth of interest or my my region of interest so what is a clean deletion clean deletion basically means you're going to take those two bracketing regions a and b here and just smush them together cleanly deleting start codon to stop
02:34
codon the orf of interest for yfg or whatever region that you want to actually delete from the genome so i call it a clean deletion this is going to be a scarless deletion it just it's as if the gene or orf doesn't even exist within the genome so that's what we're going to do that's that's the goal of kind of what i'm going to show you these techniques today again as i talk about at the end you can use these techniques for a variety of other things as well i'm going to introduce you to a plasmid i created or mark nishimura created about a decade ago we use this widely in
03:06
pseudomonads um it works in some other things too and it's we just use it really well to manipulate genomes of a lot of different gram-negative bacteria this plasmid has a narrow host range so it replicates well in e coli doesn't replicate in other things like xanthomonas or pseudomonas and so it's it's useful as a plasmid with a conditional origin replication where we can actually have it replicate e coli have e coli manipulated in e coli and then conjugate it over to other strains that where we can actually then select for the plasmid to integrate into the genome
03:36
this plasmids mtn 1907 it's got a few different features on it that i just want to point out here there's a multiple cloning site we've made a gateway compatible version but originally it was a just regular old restriction enzyme cloning version that you could use there is a gene for tet a which provides tetracycline resistance there's also a dual cassette for npt2 and sac b um mpt2 is catamycin resistance sac b is going to be for sucrose sensitivity uh the the catamycin resistance on this plasmid is a little
04:07
weird uh the promoter doesn't necessarily work in pseudomonads and so this plasmid actually provides catamycin resistance in e coli and tetracycline resistance in e coli but we can really only use it to select for tetracycline resistance um in things like pseudomonas um it's a promoter thing right so that's the layout of the plasmid now let's dive into uh the sac b gene all right so the sac b gene was originally identified in bacillus the strain of bacillus sac b stands for uh
04:37
levant sucrase it's it's a gene usually involved in an operon that manipulates sucrose and turns it into something else as we'll talk about in a second sac b is an enzyme that can take sucrase sucrose and this other long name thing over here on the left basically manipulate them change them around a little bit it turns sucrose into something called 11. 11. i'll just kind of click through here again 11 is actually toxic to many gram-negative species and so
05:09
what this gene does is it allows for uh counter selection allows for negative selection against the presence of sucrose if you grow your gram-negative bacteria on a plate with about 10 sucrose and it does this because the sac b gene will enzymatically convert sucrose into 11 11 we don't quite know how it actually kills the cells there's a little bit of mystery about that um but it's a really good it's really good at killing cells um it basically uh and we think it might get into the periplasm and kind of gunk up the works there right so
05:41
sac b is a good counter selectable marker uh so we've talked about positive selection before in terms of antibiotic resistance where you can select four genes to go into a genome a negative selection or counter selection uh is something where we can select four genes to come back out of the genome and so that's where the sac b becomes useful because we can actually use it to select for things like the plasmid to flip back out of the genome as i'll show you uh in a few slides all right so we'll run through the process from start to finish here because it's a little bit um you kind of have to wrap your mind
06:11
around some of the recombination steps here a little bit we're going to start off with our region of interest again a and b bracketing yfg we're going to make a version of this where we're going to basically mock up in dna a version of what we want the genome to look like i want to make this a clean deletion so we're going to build a synthetic piece of dna where just a and b are smushed together without yfg there you can do this through just ordering genes online now you can do it through splicing overlap pcr you can do it
06:43
through some ligations there's there's a few different ways to do this but really just it's just easiest to order these um now online all right so we've got our synthetic piece of dna made with the desired change of interest in this case the clean deletion what we're going to do is we're going to clone this into the multiple cloning site of uh 1907. so we clone that into the multiple cloning side of the 1907 and you'll just note i've i've changed the notation here on the a and b regions i've just called them a prime and b b prime to denote uh which regions
07:14
coming from the plasmid and which region is actually in the chromosome so just just take note of that all right so we've got plasmid1907 it's replicated in e coli there's an ori t on plasma 1907 so it'll actually conjugate out of e coli into a destination strain but it's only going to replicate any coli and so once it conjugates over to something like pseudomonas or xanthomonas this plasmid is not going to replicate so the only way to provide tetracycline resistance for this tet a gene to actually function is to integrate into the genome um and
07:45
be replicated as part of the genome and thereby allowing for tetracycline resistance the tet a gene to actually the protein to actually be made uh how can it actually replica integrate into the genome well we've provided all of the homologous the similar sequences it needs to actually do this this a prime region and the b prime region they're going to be sequence identical to regions on the genome where we actually want the plasmid to integrate into so all we have to do is kind of get this plasmid into the strain of interest and then let things like rec a do the work um rec a is gonna find those similar sequences
08:16
comb through the chromosome and then integrate this similar sequence into the chromosome it's gonna happen at a low frequency but you know we're dealing with microbial genetics here so we even one in a million events we can select for pretty easily sometimes all right so what does this look like um functionally well we've got our plasmid we've merged we've conjugated it into the strain of interest and now it's integrated into the genome uh it's in i've shown this here by kind of denoting both the a and a prime and b and b prime regions as where they came from
08:47
from the plasmid itself again the plasma just kind of just smushes its way into the chromosome through recombination and so it's going to be replicated as part of the chromosome and because it has that tetracycline resistance gene we can positively select for this plasmid to integrate into the genome by growing strains on tetracycline this is a positive selection for the plasmid to integrate into the genome you won't get tetracycline resistance other ways because the plasmid won't replicate in the um the strain of the destination strain of choice all right so going back to what it looks
09:20
like in the genome here we've got our plasmid 1907 replicated into the genome uh a prime a b prime b you kind of see where all the gene pieces come from and so now all we're going to do once it's in the genome the strain is going to be tetracycline resistant and it's going to be sensitive to sucrose because sac b is actually there so all we have to do is uh take the strain grow it up in non-selective media so media that doesn't have tetracycline in it and then just through again through the magic of rec a there's going to be some recombination
09:50
steps where the plasma can then recombine out of the genome uh it can recombine in two different ways as i'll show you in a second so really we're just hoping it recombines in the way we want versus kind of recapitulating the wild type sequence but really it's it's a chance thing it's going to happen one way versus the other and we just have to get lucky and screen through for the right recombination event to isolate it all right so what does this recombinant or combination event look like well if it recombines one way it'll basically recombine sequences so that the wild
10:20
type sequence the wild-type chromosomal sequence is remade um and as this is basically the plasma flips out through homologous recombination through the left a and the right a um we don't want these as baking remaking the wild type strains so you know we want to avoid this um but but it happens a lot and you just kind of have to screen through and find the one of interest um how do we actually select for the ones that don't have the plasmid there again we're going to grow this on 10 sucrose media the sucrose is going to uh select against the presence of sac b and so we're really just selecting for
10:52
strains that have flipped the plasmid out um providing you know sucrose resistance in this case and so that's recombination one way we can also recombine things the other way in the way of interest where we actually get the clean deletion and so in this case again we're it's going to happen you know in the same non-selective overnight culture as before but we're just going to try to screen through and find the recombination event of interest and what does this look like well there's going to be a homologous recombination between the left b prime and the right b prime uh and the
11:22
right b leaving the only region in the genome looking like the clean deletion as we want it so we've used recombination to flip in the plasmid um with the deletion construct into the genome and now we've used sucrose sensitivity to select against the plasmid to recombine out of the genome and now we're screening through to find recombination events that happen this way versus the wild type way so really it's just kind of putting in the putting in the work to screen enough colonies to find the one of interest that's sucrose resistant um and has the
11:53
deletion like we want it all right uh when i showed this originally i showed the plasma integrated into the genome one way um but it can actually go into the genome a couple ways uh which i'll show you one in one slide but but first i just want to highlight again that sac b is going to provide sucrose sensitivity so it allows for counter selection uh against the presence of the plasmid all right like i mentioned before um the plasmid when it integrates into the genome can integrate in a couple different ways before we showed it where it kind of broke
12:24
up the a regions of the genome the a brackets of the genome now i've just shown you an orientation here where the plasma is integrated into the b regions of the genome same deal you know it's it's just basically it can go in through recombination in any of the the similar sequence tracks that we've provided for with the a prime and the b prime sequences but in this case the plasmid has gone in and in uh b prime is recombined with b um and it's just kind of flipped around the orientation of things but this construct will work just fine for generating deletions too um you know we basically just want to
12:54
select for the tetracycline resistance to get it into the cells uh into the chromosome and then we're just hoping to to flip it out in the right way right lastly so that's the steps through to create a clean deletion this is a pretty powerful technique though so you can actually use it in a variety of other means to generate different changes in the genome as you would want so we can take our wild type gene of interest and we can actually just do allelic replacement the same way we do a clean deletion we just basically synthesize our dna
13:24
so it has a allelic replacement instead of a clean deletion as i show on the bottom there likewise we can use this as a use this construct as a landing place for completely non-homologous sequences within the genome as long as we bracket it with those a and b regions that can actually then recombine to the genome so we can use this technique and these plasmids to actually put new genes novel dna whatever we want into very specific place in the genome and then it's just a question of screening through finding the recombination event that we're looking for and then you've manipulated the genome
13:55
in the way of interest

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