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vivo expression technology


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#1 lyok

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Posted 22 July 2009 - 05:46 AM

Hallo all,

recently I have stumbled at IVET but I am not sure if I completely understand this method.

Let me write how I understand this technique.

First you mutate your bacterial strain you want to research. You generate a mutated strain that does NOT have a certain survival gene wich is needed to survive in a certain environment that you are researching.

Then you generate a plasmid wich does have this gene (= the essential survival gene)+ a gene that can be used to see if this gene is active only in vitro or also in vivo.

Then you take the original bacterial strain, "cut" this dna in pieces and insert this in the plasmid.

Then you put this plasmid in the mutated strain and then you put this "new" bacteria in the invironment you want to research, so that only those bacteria with the dna pieces that have the right promotor will survive.

Is this correct?

Then you can plate them out on some medium to check whether the promotor is active only in vivo or also in vitro.

If it is active in vitro, you do not use those or check those.


Now what is the goal of aim of this?
Is it simply a method to search for a certain promotor?

And another question: the second gene (after the essential survival gene) is used to see whether the promotor is active in vitro and in vivo or only in vivo, but this second gene, has this then the same promotor as the first one (the essential survival one). If so, are those genes then linked together so that they have the same promotor: an operon?
or?

thanks in advance

#2 fishdoc

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Posted 22 July 2009 - 06:09 AM

Hallo all,

recently I have stumbled at IVET but I am not sure if I completely understand this method.

Let me write how I understand this technique.

First you mutate your bacterial strain you want to research. You generate a mutated strain that does NOT have a certain survival gene wich is needed to survive in a certain environment that you are researching.

Then you generate a plasmid wich does have this gene (= the essential survival gene)+ a gene that can be used to see if this gene is active only in vitro or also in vivo.

Then you take the original bacterial strain, "cut" this dna in pieces and insert this in the plasmid.

Then you put this plasmid in the mutated strain and then you put this "new" bacteria in the invironment you want to research, so that only those bacteria with the dna pieces that have the right promotor will survive.

Is this correct?

Then you can plate them out on some medium to check whether the promotor is active only in vivo or also in vitro.

If it is active in vitro, you do not use those or check those.


Now what is the goal of aim of this?
Is it simply a method to search for a certain promotor?

And another question: the second gene (after the essential survival gene) is used to see whether the promotor is active in vitro and in vivo or only in vivo, but this second gene, has this then the same promotor as the first one (the essential survival one). If so, are those genes then linked together so that they have the same promotor: an operon?
or?

thanks in advance



I will qualify this answer by saying it's been a couple years since I've studied IVET, so some of the details may be incorrect. Also, there are a lot more details to the procedure than I am going into. And if I'm wrong about any of this, I hope someone will correct me.

The way you've explained it is not the way I understand it, but it may be just a communication breakdown.

From what I understand, you have a reporter gene (usually an antibiotic) in a plasmid (the 2nd gene you talk about above). That gene is promoterless. You digest your genomic DNA and insert the fragments upstream of the reporter gene. Any DNA fragment that contains an inducible promoter will allow the antibiotic gene to be expressed, thereby allowing the bacteria to survive in the presence of that antibiotic. If the bacterium doesn't survive, the insert doesn't contain a promoter that is active in those conditions.

Now, the reporter gene could also be some other type of reporter (fluorescent, metabolic, etc), and can be selected for by different media depending on what the reporter is.

The idea of IVET (and RIVET) is to determine which genes are expressed in certain conditions. It's frequently used for virulence studies to determine which genes are upregulated inside the host, thereby providing a way to identify virulence factors. That's the context of IVET that I've been taught... the virulence part. But it can be used for other types of studies as well.


Here is a review article that explains IVET http://www.pubmedcen...bmedid=12438320

Edited by fishdoc, 22 July 2009 - 06:16 AM.


#3 lyok

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Posted 22 July 2009 - 07:01 AM

Ok,

I see what you mean, but the promotor of this reporter gene is also the same as the for the "survival" gene (the first one) not?

But the important one you are researching is still the "survival" gene right?

But when you notice that the bacteria is surviving in the presence of that antibiotica on your agar, then you simply know that this reporter gene is active in vitro , but what about the bacteria that die?
Those can have an active promotor too... but one that is only active in vivo and not on your agar plates.

Its getting confusing now lol.

#4 fishdoc

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Posted 22 July 2009 - 07:16 AM

Ok,

I see what you mean, but the promotor of this reporter gene is also the same as the for the "survival" gene (the first one) not?

But the important one you are researching is still the "survival" gene right?

But when you notice that the bacteria is surviving in the presence of that antibiotica on your agar, then you simply know that this reporter gene is active in vitro , but what about the bacteria that die?
Those can have an active promotor too... but one that is only active in vivo and not on your agar plates.

Its getting confusing now lol.



If you can, read that article. It's basically how I learned about IVET.

After my initial response, I went back and read up on the procedure and have a better idea of it now.

I think in the case you're talking about, say you have a bacterial strain that is deficient for purA, so it has a problem making purines. Your "survival" gene on your plasmid then is purA to complement the mutation in the bacterial genome. That purA will be promoterless. So you insert your digested genomic pieces into the multiple cloning site of the plasmid, upstream of the promoterless purA. The purA mutants carrying the constructs are then put into whatever animal you're talking about, and the only ones that survive coming out of the animal are the ones that have a piece of DNA inserted in the MCS that has an inducible promoter that activates purA on the plasmid to complement the purA mutation in the genome. I THINK the second gene, which would be a reporter gene, does not have its own promoter, because it is used for selection that the inserted sequence does not contain a promoter that is active ex vivo, but I'm NOT for sure on that.

The purA gene can also be an antibiotic resistance gene, but if that's the case your animal will need to have been treated with that antibiotic in order to select for the plasmids carrying the resistance cassette.

Basically, the purA or resistance gene allows for survival inside the animal. The 2nd gene, like lacZ, provides for screening ex vivo to be sure the inserted sequence is only active in vivo. So either before you infect your animal, or when you retrieve them from the animal (or both), you plate on S-Gal or X-Gal, and select white colonies only. If the colonies are blue or black, the lacZ is being made, which means the promoter is active ex vivo.

IVET is used to determine what promoters, and therefore what genes are active in vivo only.

So the promoter operates both the auxotrophic gene/resistance gene (gene 1) and the reporter gene (gene 2).

Basically, what you're researching are the inserts you put into the MCS of the two genes, the two genes are simply tools to use to identify in vivo-induced genes.


If you're still confused, perhaps you could describe the structure of the plasmid you're working with, which gene is #1, which is #2, etc.

Edited by fishdoc, 22 July 2009 - 07:18 AM.


#5 lucilius

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Posted 23 July 2009 - 04:17 AM

Very interesting topic since I am also reading on this topic.

However I do have a question: when they create the plasmid with the degisted DNA in it(with hoppefull the right promotor), do they use the same restriction enzyme to cut the plasmid open and to digest the dna that you want to insert in the plasmid?

Or do they cut the DNA at random with several restriction enzymes?

I am speaking about the first 2 steps : digesting the DNA you want to insert in the plasmid and the "opening" of the plasmid you will be using to get your dna in the mutated strain.

To me it seems logical they need to use the same restriction enzyme to cut both the DNA and the plasmid so that they can fit into eachother.


Another basic question: the DNA that is digested is this the DNA of the NOT mutated strain? Meaning that this DNA can have the essential growth factor gene + the promotor of this one? ==> if so is then not possible you will insert a piece of dna in your plasmid that contains both the promotor you look for AND the essential growth factor gene?

Or is this not possible because you cut this DNA into very small pieces?

Or is this the DNA of the mutated strain? meaning it has no longer the essential growing factor?


thanks

#6 fishdoc

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Posted 23 July 2009 - 04:32 AM

Very interesting topic since I am also reading on this topic.

However I do have a question: when they create the plasmid with the degisted DNA in it(with hoppefull the right promotor), do they use the same restriction enzyme to cut the plasmid open and to digest the dna that you want to insert in the plasmid?

Or do they cut the DNA at random with several restriction enzymes?

I am speaking about the first 2 steps : digesting the DNA you want to insert in the plasmid and the "opening" of the plasmid you will be using to get your dna in the mutated strain.

To me it seems logical they need to use the same restriction enzyme to cut both the DNA and the plasmid so that they can fit into eachother.


Another basic question: the DNA that is digested is this the DNA of the NOT mutated strain? Meaning that this DNA can have the essential growth factor gene + the promotor of this one? ==> if so is then not possible you will insert a piece of dna in your plasmid that contains both the promotor you look for AND the essential growth factor gene?

Or is this not possible because you cut this DNA into very small pieces?

Or is this the DNA of the mutated strain? meaning it has no longer the essential growing factor?


thanks



I've never done IVET, but I'd assume you use one enzyme to digest the genome and the plasmid.

For the genomic to be digested, I don't think it matters much. For the mutant gene, you already know what it does, or else it wouldn't be mutated and complemented. And if you did use wild type genome, when you screen the colonies in vitro either before or after putting them in vivo, you'd get blue colonies (if using lacZ as a reporter) because it would be active ex vivo. You then would not include that isolate in the analysis because it's not one induced in vivo.

#7 lucilius

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Posted 23 July 2009 - 04:42 AM

Ok, I see.

I do have one "strange" question maybe, but what it the point of finding the right promotor?

They always say IVET is a promotor trapping method since you get bacteria with the right promotor (they are the only one that survives), but what do you do with those bacteria containing that promotor?
Do you then search for the dna sequence of that promotor so you know the dna sequence of that promotor? Or ?
(I can not imagine you start looking for this promotor sequence, since you would need to know all the other genes in your bacteria and if you allready know those, you also know that promotor:s)

Or is it just a way to make bacteria having that promotor and then use those? But then again: your not mutated strain, allready has this promotor so why "recreate" this strain?

#8 fishdoc

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Posted 23 July 2009 - 07:03 AM

Ok, I see.

I do have one "strange" question maybe, but what it the point of finding the right promotor?

They always say IVET is a promotor trapping method since you get bacteria with the right promotor (they are the only one that survives), but what do you do with those bacteria containing that promotor?
Do you then search for the dna sequence of that promotor so you know the dna sequence of that promotor? Or ?
(I can not imagine you start looking for this promotor sequence, since you would need to know all the other genes in your bacteria and if you allready know those, you also know that promotor:s)

Or is it just a way to make bacteria having that promotor and then use those? But then again: your not mutated strain, allready has this promotor so why "recreate" this strain?



IVET is a method to determine in vivo-induced genes. Once you have promoters that are activated in vivo, you have an idea of genes that are induced in vivo. This is commonly used for virulence genes (there may be other applications, but none that I can think of). It's basically just a way to screen a bunch of promoters to find out which ones are induced in vivo. Then once you know the promoters that are induced, you can look at the genes associated with those promoters to determine genes that are turned on in vivo, which are likely to be involved in virulence.

You may know all the genes in your genome, but you don't know which ones are induced where, which is where the IVET comes in.

#9 lucilius

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Posted 23 July 2009 - 07:07 AM

Oh ok.

I see.
I had a complete wrong general idea then.

#10 fishdoc

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Posted 23 July 2009 - 07:11 AM

Oh ok.

I see.
I had a complete wrong general idea then.



You may or you may not have. Bacterial pathogenesis is the context under which I learned IVET. There may be other applications to it that I haven't read about.

#11 lucilius

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Posted 23 July 2009 - 07:13 AM

Eum, oh well, as long as I have a better view on it, its ok.

I do have one more question, but maybe I better start a new topic about this.

Anyway: how do they create those mutated bacteria strains that have a gene deletion.

I do not see how you can simply delete a gene from a bacteria.

I have read something about the use of plasmids to do this, but how does this work?

#12 fishdoc

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Posted 23 July 2009 - 07:22 AM

Eum, oh well, as long as I have a better view on it, its ok.

I do have one more question, but maybe I better start a new topic about this.

Anyway: how do they create those mutated bacteria strains that have a gene deletion.

I do not see how you can simply delete a gene from a bacteria.

I have read something about the use of plasmids to do this, but how does this work?



It's very easy and done quite often.

It can be done with plasmids or bacteriophage.

The way I've done it is to either create a deletion in the sequence by PCR or restriction digest. Alternatively, you can put an antibiotic resistance marker in the middle of a gene to disrupt it. Then, the construct gets subcloned into a suicide plasmid and conjugated into the wild type bacteria. Once inside the plasmid can't replicate (hence the name "suicide vector"), and it eventually disappears unless it is integrated. Integration can occur via the gene-sequence in the plasmid recombining with the genomic DNA, and allelic exchange occurs. The mutation goes into the genome, and you select for it either on antibiotic resistance, metabolic dependency, or some other phenotype.

This is a very brief and watered down explanation of how to do it, but it covers the basics. There are numerous papers out there detailing bacterial mutagenesis, and there may be some articles in the "protocols" link at the top of this page.

This topic is large, and probably isn't suited for a message board (at least the theory or practice of bacterial mutagenesis), so you'd probably be better off finding some literature or talking to someone in person about how it's done.

I would be happy to answer specific questions about the process, but can't give a detailed explanation of the entire process.

#13 lucilius

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Posted 23 July 2009 - 10:48 AM

Yeah, you are right.
The problem is that I do not have a good molecular biology background, so its hard for me to understand the scientif explenations used in the articles.

Integration can occur via the gene-sequence in the plasmid recombining with the genomic DNA, and allelic exchange occurs. The mutation goes into the genome, and you select for it either on antibiotic resistance, metabolic dependency, or some other phenotype

2 more questions about this: how does the dna from the plasmid get into the genome of the bacteria? Integration, ok, but what is this? How can it get in the chromosome of the bacteria?


You say that the plasmid will die if it is not integrated in the genome (since its a suicide vector), but to get integrated , does the plasmide fall apart? The wall of the plasmid must break down first to set the dna free or?

(PS. by suicide plasmid you do mean a plasmid without a Origin of replication? or not?)

Edited by lucilius, 23 July 2009 - 10:53 AM.


#14 fishdoc

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Posted 23 July 2009 - 11:11 AM

Yeah, you are right.
The problem is that I do not have a good molecular biology background, so its hard for me to understand the scientif explenations used in the articles.

Integration can occur via the gene-sequence in the plasmid recombining with the genomic DNA, and allelic exchange occurs. The mutation goes into the genome, and you select for it either on antibiotic resistance, metabolic dependency, or some other phenotype

2 more questions about this: how does the dna from the plasmid get into the genome of the bacteria? Integration, ok, but what is this? How can it get in the chromosome of the bacteria?


You say that the plasmid will die if it is not integrated in the genome (since its a suicide vector), but to get integrated , does the plasmide fall apart? The wall of the plasmid must break down first to set the dna free or?

(PS. by suicide plasmid you do mean a plasmid without a Origin of replication? or not?)



The plasmid has an origin of replication that is dependent upon a certain protein. If that protein is not present, the plasmid does not replicate, and eventually gets diluted out. You propagate the plasmid in an E. coli strain that produces the needed protein (lambda pir is an example). But when it's conjugated or electroporated into the target bacterium, it doesn't replicate if that bacterium doesn't encode the protein.

The plasmid DNA enters the genome by homologous recombination. An explanation of that should be available in any bacterial genetics text, and I'd imagine there are explanations available online for that process.

#15 lucilius

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Posted 23 July 2009 - 12:47 PM

Thanks again for the answer.

And about homologous recombination: I have checked it, and know it, however it is always explained with 2 ds-DNA ; = + = ==> ..)



However I am also looking into it in the case of 1 ds dna (the initial ds dna of the bacteria and the new single stranded dna you insert , because I am also interested in the insertion of naked dna , not using a plasmid)
And about this I only seem to find very basic information like: the single straned dna enters the cel and is then integrated in the original chromosome by homologous recombination.
And then I always stumble back at the part where you have 2 ds dna pieces, here you onyl have 1 , so I wonder how you get that 1 piece in or on the double stranded piece of dna.




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