suicide vector - what is this? (Aug/23/2009 )
Hallo all,
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
-lucilius-
lucilius on Aug 24 2009, 04:06 AM said:
Hallo all,
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
A suicide vector is one that will kill a host cell only if it does not have an inserted sequence. Typically, the cloning region is within the suicide gene. If the plasmid has not successfully taken up an insert, ie recircularised, the gene is expressed and the cell dies. If, on the other hand, a gene is ligated in, the suicide gene is interrupted and the cell lives. It is, therefore, a way to reduce background.
Homologous recombination doesn't happen during replication, so the problem of single- versus double-stranded DNA disappears.
Regarding low copy number plasmids, you're absolutely correct. Some plasmids are present in hundreds of copies per cell, others only have 1 or 2.
-swanny-
swanny on Aug 23 2009, 06:08 PM said:
lucilius on Aug 24 2009, 04:06 AM said:
Hallo all,
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
A suicide vector is one that will kill a host cell only if it does not have an inserted sequence. Typically, the cloning region is within the suicide gene. If the plasmid has not successfully taken up an insert, ie recircularised, the gene is expressed and the cell dies. If, on the other hand, a gene is ligated in, the suicide gene is interrupted and the cell lives. It is, therefore, a way to reduce background.
Homologous recombination doesn't happen during replication, so the problem of single- versus double-stranded DNA disappears.
Regarding low copy number plasmids, you're absolutely correct. Some plasmids are present in hundreds of copies per cell, others only have 1 or 2.
I've understood a suicide vector to be something a little different. Rather than it killing the cell, it cannot replicate without a certain protein, which is usually provided by the host strain.
We use pGP704, which has always been referred to as a suicide vector, to transfer mutations by homologous recombination. pGP704 does not replicate except in the presence of the pir protein. A few strains of E. coli we have encode this protein (CC118, SM10, S17-1).
Basically how it works is we clone a sequence of DNA containing a gene mutation into pGP704 (and more recently a pGP704 derivative, pRE107), and then conjugate it to our species of study using SM10 lambda-pir. Because our bacterial species does not encode lambda pir, the vector does not replicate. For the most part, the vector will just disappear by dilution as the cells replicate, but some will integrate into the genome via the homologous DNA sequences (the 5' and 3' regions flanking the mutation site). There can either be a single crossover event which results in the integration of the entire plasmid, or there can be a double crossover event resulting in an exchange of the native DNA sequence with the mutated sequence, thus resulting in the desired mutation in the genome.
Like I said, that's how I've understood a suicide vector, but perhaps it isn't correct.
As for your questions about how recombination would work, I can't explain it in a post. There's too much involved. There should be plenty of information available on the web or in a good microbial genetics textbook. RecA, Holliday structures, all that good stuff.
-fishdoc-
fishdoc on Aug 24 2009, 10:40 AM said:
swanny on Aug 23 2009, 06:08 PM said:
lucilius on Aug 24 2009, 04:06 AM said:
Hallo all,
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
I was wondering about what a suicide vector is.
As I understand it, it is a vector (plasmid) that is unable to reproduce itself in the host?
But what does this mean? Does this mean that this vector, plasmid will die if the host doesnt integrate it in his own chromosomal dna?
And how does the host integrate this plasmid dna anyway?
I have read about homologous recombination, but I can not see how this would work since the dna of the host itself is singlestranded when being copied, but the dna of the plasmid stays doublestranded, so how can there be homologous recombination if the dna of that plasmid stays doublestranded?
(I can not see that a singlestranded piece of host dna that is being reproduced would react with double stranded dna of that plasmid).
And is this suicide vector then called an episome (when it does integrate in the hosts dna)?
Or not?
And 1 more question.
A low copy vector, does this simply mean that the vector does reproduce itself , but at very low numbers? Meaning that the number of plasmids in the cell will not get high ? Or ?
A suicide vector is one that will kill a host cell only if it does not have an inserted sequence. Typically, the cloning region is within the suicide gene. If the plasmid has not successfully taken up an insert, ie recircularised, the gene is expressed and the cell dies. If, on the other hand, a gene is ligated in, the suicide gene is interrupted and the cell lives. It is, therefore, a way to reduce background.
Homologous recombination doesn't happen during replication, so the problem of single- versus double-stranded DNA disappears.
Regarding low copy number plasmids, you're absolutely correct. Some plasmids are present in hundreds of copies per cell, others only have 1 or 2.
I've understood a suicide vector to be something a little different. Rather than it killing the cell, it cannot replicate without a certain protein, which is usually provided by the host strain.
We use pGP704, which has always been referred to as a suicide vector, to transfer mutations by homologous recombination. pGP704 does not replicate except in the presence of the pir protein. A few strains of E. coli we have encode this protein (CC118, SM10, S17-1).
Basically how it works is we clone a sequence of DNA containing a gene mutation into pGP704 (and more recently a pGP704 derivative, pRE107), and then conjugate it to our species of study using SM10 lambda-pir. Because our bacterial species does not encode lambda pir, the vector does not replicate. For the most part, the vector will just disappear by dilution as the cells replicate, but some will integrate into the genome via the homologous DNA sequences (the 5' and 3' regions flanking the mutation site). There can either be a single crossover event which results in the integration of the entire plasmid, or there can be a double crossover event resulting in an exchange of the native DNA sequence with the mutated sequence, thus resulting in the desired mutation in the genome.
Like I said, that's how I've understood a suicide vector, but perhaps it isn't correct.
As for your questions about how recombination would work, I can't explain it in a post. There's too much involved. There should be plenty of information available on the web or in a good microbial genetics textbook. RecA, Holliday structures, all that good stuff.
Fishdoc, I think you are right. I was thinking about one particular vector (from mobiotec, I think) that does what I described.
-swanny-
Homologous recombination doesn't happen during replication, so the problem of single- versus double-stranded DNA disappears.
Oh ok, I see, that does indeed change the whole idea behind it.
As for your questions about how recombination would work, I can't explain it in a post. There's too much involved. There should be plenty of information available on the web or in a good microbial genetics textbook. RecA, Holliday structures, all that good stuff.
I have tried to look it up and understand it, but I am still confused because they never explain why the plasmids dna is involved in it.
Because the mechanism is always talking about "working" with the dna of the cell itself (the genomic dna), but how does the plamids dna get involved in it.. thats what I do not completely see.
Because during the recombination you have that RecA that is working with the genomic dna, its cutting there, but how does it get on the plasmids dna then? its just "by accident" then, because its homologou?
-lucilius-
lucilius on Aug 24 2009, 04:09 AM said:
Homologous recombination doesn't happen during replication, so the problem of single- versus double-stranded DNA disappears.
Oh ok, I see, that does indeed change the whole idea behind it.
As for your questions about how recombination would work, I can't explain it in a post. There's too much involved. There should be plenty of information available on the web or in a good microbial genetics textbook. RecA, Holliday structures, all that good stuff.
I have tried to look it up and understand it, but I am still confused because they never explain why the plasmids dna is involved in it.
Because the mechanism is always talking about "working" with the dna of the cell itself (the genomic dna), but how does the plamids dna get involved in it.. thats what I do not completely see.
Because during the recombination you have that RecA that is working with the genomic dna, its cutting there, but how does it get on the plasmids dna then? its just "by accident" then, because its homologou?
I'm not quite how to explain it all. In homologous recombination, there are two DNA sequences involved, the donor and the recipient. In this case, the plasmid is the donor and the genome is the recipient. Based on any sort of graphic illustrating homologous recombination, it should work from there. I think we talked about this stuff in the IVET discussion awhile back, with a few images.
-fishdoc-
Bit difficult to explain it here...
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-lovely01-
lovely01 on Aug 26 2009, 01:34 PM said:
Bit difficult to explain it here...
How do you mean?
-lucilius-
lucilius on Aug 26 2009, 10:13 AM said:
lovely01 on Aug 26 2009, 01:34 PM said:
Bit difficult to explain it here...
How do you mean?
I can't speak for Lovely, but for me, I had a microbial genetics class, and it was a good couple of lectures (if not more) just dealing with the ins and outs of homologous recombination, enzymes involved, etc. Something like that cannot be packed into a message board post. It either needs to be addressed in a class, or by learning it from a text. A message board cannot do it justice.
-fishdoc-
fishdoc on Aug 26 2009, 07:16 PM said:
lucilius on Aug 26 2009, 10:13 AM said:
lovely01 on Aug 26 2009, 01:34 PM said:
Bit difficult to explain it here...
How do you mean?
I can't speak for Lovely, but for me, I had a microbial genetics class, and it was a good couple of lectures (if not more) just dealing with the ins and outs of homologous recombination, enzymes involved, etc. Something like that cannot be packed into a message board post. It either needs to be addressed in a class, or by learning it from a text. A message board cannot do it justice.
I see your point.
Anyway, thanks to you, I allready understand +-80% of the problem, however there are still a few little details that I do not completly understand.
But maybe you are right and it can only be explained in person.
You say something about a good text to learn it, but I have not yet found a real good one that answers all my questions on a simple or efficient, clear way.
-lucilius-