We are trying to generate deletion mutants using a sacB counterselection system. We have successfully transformed the sacB plasmid with flanking regions of our gene or interest into the recipient strain and confirmed this by antibiotic resistance. Sucrose counterselection has also been very efficient, and loss of drug resistance has been confirmed (~50%). However, when we test the newly sensitive candidates (~30) none of them have the deletion mutations; all have reverted back to wild type. Does anybody have a good explanation for this? Have any of you experienced something similar and do you have any advice for troubleshooting? Thank you!
It is really hard to diagnose with the information you provided. Are you certain you have designed the SacB plasmid correctly? (A drawing might help). Are the flanking regions of homology comparable in size?
Thanks for replying. I'm attaching files of the plasmid - flanking regions are color-coded pink (5'; 360 bp upstream of the gene of interest + 163 bp of the gene) and purple (3'; 210 bp of the gene, 589 downstream flank). So yes, they are roughly the same size. I included the start and end of the gene to ensure an in-frame deletion as this is the first gene in a large operon.
I interpreted the results as only giving us WT revertants, rather than the correct deletion, after the sucrose counterselection step. It sounds like maybe we aren't even getting that far, although if we are seeing sucrose counterselection, the plasmid must be integrated into the chromosome, right? It is a suicide plasmid after all. Nonetheless, the next go around we will screen transformants by PCR as well as check for drug resistance. So maybe the plasmid is integrating into a different location other than the desired one.
If the flanking regions are the same size then you should get 50% wt and 50% deletion assuming everything is working well.
Did you obtain a few independent integrants and try to cure them separately, you might just have an anomalous integration event. That would be the first thing I would try.
You could confirm that your plasmid is truly integrated where it is supposed to be, use one primer just upstream of your upstream flanking region (so that this primer is not present anywhere on the plasmid) and then one primer from within the plasmid. But be aware that there are two different possible integrants (one with the deletion upstream and one with the intact gene upstream) of the integrated plasmid.
We had similar experience of the mutations reverting back to wild type if you passage the transformants in the cultures. We could manage to continue with analysis of the transformants by making new transformations when we had to do analyses.
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