12 replies to this topic

[lyok]

Dear all,

I have the following question: I have a bacterium with a certain geneX that I want to remove. I only have a plasmid that has the same geneX in his DNA. Now I have no clue how to remove the geneX from the chromosomal DNA of my bacterium.

According to someone it is possible to remove a gene from DNA with the use of a plasmid that has a homologous DNA sequence, however I can not figure this out, I do not understand how I can remove a gene when only using a plasmid that has the same gene. When using homologous recombination this would then not remove the gene but simply double it ?
Or am I missing something here?

The only thing I can imagine is to ligate another genecluster inside the plasmids geneX (the one I want to remove from the DNA). So that the geneX in the bacterium will be “ruined” with homologous recombination.
However the geneX isn’t removed from the chromosomal DNA then, I wonder if I can completely remove the geneX with the use of the plasmid that holds a identical geneX?

Another idea would be to remove the geneX from the DNA by inserting the same genes that are flanking this geneX in the chromosomal DNA in the plamid so that I can remove it from the DNA with homologous recombination, however I do not know the sequence of the genes before or after the geneX I want to remove, so this is not an option.

Anyone an idea?

[fishdoc]

lyok, on Nov 16 2009, 09:25 AM, said:

Dear all,

I have the following question: I have a bacterium with a certain geneX that I want to remove. I only have a plasmid that has the same geneX in his DNA. Now I have no clue how to remove the geneX from the chromosomal DNA of my bacterium.

According to someone it is possible to remove a gene from DNA with the use of a plasmid that has a homologous DNA sequence, however I can not figure this out, I do not understand how I can remove a gene when only using a plasmid that has the same gene. When using homologous recombination this would then not remove the gene but simply double it ?
Or am I missing something here?

The only thing I can imagine is to ligate another genecluster inside the plasmids geneX (the one I want to remove from the DNA). So that the geneX in the bacterium will be “ruined” with homologous recombination.
However the geneX isn’t removed from the chromosomal DNA then, I wonder if I can completely remove the geneX with the use of the plasmid that holds a identical geneX?

Another idea would be to remove the geneX from the DNA by inserting the same genes that are flanking this geneX in the chromosomal DNA in the plamid so that I can remove it from the DNA with homologous recombination, however I do not know the sequence of the genes before or after the geneX I want to remove, so this is not an option.

Anyone an idea?

Delete a region of geneX in the plasmid, either by inverse PCR, restriction digest, or some other method. Then insert that construct into a suicide vector and transform E. coli with it. Homologous recombination will then insert the deletion construct in place of the native allele. However, in order to achieve homologous recombination, you will need an adequate amount of DNA flanking the deletion. That amount depends on a few things, like if the lambda red phage is present. If it is, you need as little as about 40 bp of flanking DNA. If that's not present, it's best to use at least 500 bp of flanking sequence (in my experience).

The other option is to find a restriction site in geneX in the plasmid and insert a antibiotic resistance cassette. That will disrupt the gene, but will not delete it. Either way, it should knock out expression.

[lyok]

fishdoc, on Nov 16 2009, 06:48 PM, said:

lyok, on Nov 16 2009, 09:25 AM, said:

Dear all,

I have the following question: I have a bacterium with a certain geneX that I want to remove. I only have a plasmid that has the same geneX in his DNA. Now I have no clue how to remove the geneX from the chromosomal DNA of my bacterium.

According to someone it is possible to remove a gene from DNA with the use of a plasmid that has a homologous DNA sequence, however I can not figure this out, I do not understand how I can remove a gene when only using a plasmid that has the same gene. When using homologous recombination this would then not remove the gene but simply double it ?
Or am I missing something here?

The only thing I can imagine is to ligate another genecluster inside the plasmids geneX (the one I want to remove from the DNA). So that the geneX in the bacterium will be “ruined” with homologous recombination.
However the geneX isn’t removed from the chromosomal DNA then, I wonder if I can completely remove the geneX with the use of the plasmid that holds a identical geneX?

Another idea would be to remove the geneX from the DNA by inserting the same genes that are flanking this geneX in the chromosomal DNA in the plamid so that I can remove it from the DNA with homologous recombination, however I do not know the sequence of the genes before or after the geneX I want to remove, so this is not an option.

Anyone an idea?

Delete a region of geneX in the plasmid, either by inverse PCR, restriction digest, or some other method. Then insert that construct into a suicide vector and transform E. coli with it. Homologous recombination will then insert the deletion construct in place of the native allele. However, in order to achieve homologous recombination, you will need an adequate amount of DNA flanking the deletion. That amount depends on a few things, like if the lambda red phage is present. If it is, you need as little as about 40 bp of flanking DNA. If that's not present, it's best to use at least 500 bp of flanking sequence (in my experience).

The other option is to find a restriction site in geneX in the plasmid and insert a antibiotic resistance cassette. That will disrupt the gene, but will not delete it. Either way, it should knock out expression.

Fishdoc,

however I have one more question: if I use the "delete" method, I will insert the native geneX with a deletion, but I would not completely remove it then, is this right?

[aimikins]

well, what's your purpose?

with either method you'll retain flanking DNA, but if you just want to kick function you're covered. why would it be necessary to completely remove all traces?

[fishdoc]

lyok, on Nov 20 2009, 01:06 AM, said:

Fishdoc,

however I have one more question: if I use the "delete" method, I will insert the native geneX with a deletion, but I would not completely remove it then, is this right?

Depends on what your flanking sequence is that you use for homologous recombination. If it is outside of the gene's start and stop, the complete gene will be deleted. If the flanking sequence includes the start and stop, it won't be a complete deletion, but functionally, if you get rid of a large portion of a gene, it will be nonfunctional.

[lyok]

fishdoc, on Nov 21 2009, 12:44 AM, said:

lyok, on Nov 20 2009, 01:06 AM, said:

Fishdoc,

however I have one more question: if I use the "delete" method, I will insert the native geneX with a deletion, but I would not completely remove it then, is this right?

Depends on what your flanking sequence is that you use for homologous recombination. If it is outside of the gene's start and stop, the complete gene will be deleted. If the flanking sequence includes the start and stop, it won't be a complete deletion, but functionally, if you get rid of a large portion of a gene, it will be nonfunctional.

Ah yes, indeed.

But I dont know a lot about the plasmid. All I know is that it has a homologous region.
So I have to use your previous tips and insert something in that region (in the genex) so I can disrupt this gene in the chromosomal DNA.

@aimikins, I wanted to completely delete this geneX, but you are right: it doesnt really matter if its completely gone or disrupted.

thanks guys

[fishdoc]

lyok, on Nov 21 2009, 11:42 AM, said:

fishdoc, on Nov 21 2009, 12:44 AM, said:

lyok, on Nov 20 2009, 01:06 AM, said:

Fishdoc,

however I have one more question: if I use the "delete" method, I will insert the native geneX with a deletion, but I would not completely remove it then, is this right?

Depends on what your flanking sequence is that you use for homologous recombination. If it is outside of the gene's start and stop, the complete gene will be deleted. If the flanking sequence includes the start and stop, it won't be a complete deletion, but functionally, if you get rid of a large portion of a gene, it will be nonfunctional.

Ah yes, indeed.

But I dont know a lot about the plasmid. All I know is that it has a homologous region.
So I have to use your previous tips and insert something in that region (in the genex) so I can disrupt this gene in the chromosomal DNA.

@aimikins, I wanted to completely delete this geneX, but you are right: it doesnt really matter if its completely gone or disrupted.

thanks guys

Be mindful of whether or not it is in an operon. If it is, mutation could result in a polar effect, and an observed phenotype may not be directly related to mutation of "geneX", but rather disruption of expression of the rest of the operon. If the gene is not in an operon, you don't have to worry about that.

[fishdoc]

lyok, on Nov 21 2009, 11:42 AM, said:

But I dont know a lot about the plasmid. All I know is that it has a homologous region.

Sequence it. Know what you're working with before you work with it. It may save a lot of headaches down the road. Don't assume anything. Depending on how the construct is made, it may not even be a 100% accurate sequence... if it was cloned from a PCR product, there's a chance that amplification introduced an error you wouldn't expect.

[HomeBrew]

I agree with fishdoc -- sequence the insert of your plasmid. If the entire gene is not there, you may be able to use it directly to interrupt the gene -- the plasmid will cross into the chromosome and disrupt the gene, creating an insertional mutant, much like a transposon hit, but the insertion is targeted to your gene via the homologous region.

[pDNA]

If you want to delete a section of the genome completely you will have to use the "scarless engineering" approach published by Feher et al.. If you are interested read it up or if something is unclear to you ask again!

Regards,
p

[lyok]

pDNA, on Nov 22 2009, 08:50 AM, said:

If you want to delete a section of the genome completely you will have to use the "scarless engineering" approach published by Feher et al.. If you are interested read it up or if something is unclear to you ask again!

Regards,
p

I cant acces that file

@fishdoc and Homebrew, at the moment I can not sequence it, so I have to deal with it as it is.

[fishdoc]

lyok, on Nov 23 2009, 09:29 AM, said:

@fishdoc and Homebrew, at the moment I can not sequence it, so I have to deal with it as it is.

So you're going to attempt to make a mutant using a plasmid of unknown sequence? How will you know that you've actually made the correct mutation then? And in the event you do get a mutation made, you will have to sequence that to verify the correct sequence was manipulated. Based on my experience with bacterial mutagenesis, I will recommend strongly against going any further without verifying that plasmid sequence. If you don't know the sequence of the plasmid, you won't know what it does to the sequence of the genome. Without a genotype, any resulting phenotype is pretty worthless, because you can't make any direct correlation to the genotype you think you have. In other words, there is no shot of getting anything published, patented, or whatever you want to do with sequencing that plasmid. If you are by chance in a graduate program, there's no way that should even get by your committee. That's a major snag, in other words.

[lyok]

fishdoc, on Nov 23 2009, 05:23 PM, said:

lyok, on Nov 23 2009, 09:29 AM, said:

@fishdoc and Homebrew, at the moment I can not sequence it, so I have to deal with it as it is.

So you're going to attempt to make a mutant using a plasmid of unknown sequence? How will you know that you've actually made the correct mutation then? And in the event you do get a mutation made, you will have to sequence that to verify the correct sequence was manipulated. Based on my experience with bacterial mutagenesis, I will recommend strongly against going any further without verifying that plasmid sequence. If you don't know the sequence of the plasmid, you won't know what it does to the sequence of the genome. Without a genotype, any resulting phenotype is pretty worthless, because you can't make any direct correlation to the genotype you think you have. In other words, there is no shot of getting anything published, patented, or whatever you want to do with sequencing that plasmid. If you are by chance in a graduate program, there's no way that should even get by your committee. That's a major snag, in other words.

You are 100% correct.
But at the moment I have to work with it as it is. The plasmid is being sequenced, so for the future thats no problem.
But at the moment we are working with it as it is.

thanks for the help and pDNA thank you for the paper.