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How to exchange antibiotic resistance markers on a plasmid for E. coli


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#1 amano

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Posted 16 June 2009 - 06:02 AM

Hello,

Has anyone exchanged or heard of someone exchanging the antibiotic resistance marker on a plasmid for E. coli? I need to co-transform E coli with two plasmids that have the same resistance marker, and the plasmids are not available with any other marker. I have plasmid sequence information for the two plasmids of interest and a 3rd plasmid with the new resistance marker that identifies the location of each marker. I've confirmed with a BLAST search that the annotated sequences are simply the coding sequences for each marker (start to stop codon). Is it simply a modular procedure where I remove one ORF and replace it with the other? Or are there other considerations I need to think about?

The plasmid map doesn't annotate any promoter or terminator sequence for the resistance marker. But I assume if one is necessary, then I am only substituting the coding sequence and the remaining un-annotated architecture is still present. Or would they be marker specific?

For what it's worth, I'd like to substitute a chloramphenicol resistance marker with a kanamycin marker.

Thanks in advance for your help.

#2 eldon

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Posted 19 June 2009 - 05:30 AM

Hello,

Has anyone exchanged or heard of someone exchanging the antibiotic resistance marker on a plasmid for E. coli? I need to co-transform E coli with two plasmids that have the same resistance marker, and the plasmids are not available with any other marker. I have plasmid sequence information for the two plasmids of interest and a 3rd plasmid with the new resistance marker that identifies the location of each marker. I've confirmed with a BLAST search that the annotated sequences are simply the coding sequences for each marker (start to stop codon). Is it simply a modular procedure where I remove one ORF and replace it with the other? Or are there other considerations I need to think about?

The plasmid map doesn't annotate any promoter or terminator sequence for the resistance marker. But I assume if one is necessary, then I am only substituting the coding sequence and the remaining un-annotated architecture is still present. Or would they be marker specific?

For what it's worth, I'd like to substitute a chloramphenicol resistance marker with a kanamycin marker.

Thanks in advance for your help.


Most simple route -> look in the catalog of your fave plasmid supplier and order a cloning vector with dual selection for $50 and save yourself the hassle of re-inventing the wheel

#3 warsel

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Posted 22 June 2009 - 06:27 AM

exchanging resistance markers is probably most easily done by just switching to another empty vector with kanamycin resistance and cloning your gene of interest into that vector.

other than that you can do PCR-cloning to get it done, but you still need to know where your chloramphenicol resistance gene is on the vector in order to delete it, and you need a vector with kanamycin resistance to clone the kanamycin gene from..




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