How to exchange antibiotic resistance markers on plasmids for E coli

David Liu via methods%40net.bio.net (by amanosz from yahoo.com)
Tue Jun 16 08:09:18 EST 2009


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.

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

Thanks in advance for your help,

David




      



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