Im currently doing gene deletion. I am following the procedure in openwetware. During my first 3 gene deletion I got high efficiency, however, in my 4th deletion now, I got very very low efficiency. I only got 1 positive out of 10 colonies that I picked from the plate with antibiotic. My host strain will now have its 4th gene to be deleted.
Now I got so many background colonies. I initially suspect that the plasmid template during PCR could have caused the resistance, however, it was not the case. Now, I suspect that the background colonies were due to random insertion of my deletion cassette somewhere in the genome.
If this is the case, how can you determine whether your positive transformant only contain one insertion/recombination of the deletion cassette?
Hi there,
Are you sure the succesive deletions don't result in a lethal phenotype at the end which could explain the fact that the expected event becomes more and more unlikely? About multiple insertions : this is less frequent than single insertion therefore if you manage to show cassette insertion at the right place in the genome it's OK. You don't really care about other insertions, do you? The only problem remains frequency of the expected event. As I wrote earlier if the cassette insertion is lethal for the cell, you won't observe any positive clone...
Thank you for your answer. The deletion cassette is not lethal for the cell because many studies have done that as well. I'm just concern with my efficiency.
Individually your deletions might have no effect on cell growth but what I mean is that maybe these deletions might have a cumulative effect being deleterious to the cell viability at the end.
Have you added any antibiotic resistance marker to screen for mutants? That should reduce your background colonies.
Hi there..
You need to check the expression of deleted genes. If the gene has been deleted, its protein should not be there in the organism.
Select for isogenic colonies to get pure mutants by streaking them 3 generations and selecting them on antibiotic containing plates. I agree completely with Gaurav. You can do PCR to amplify the gene of interest. Use a positive control/WT where it will amplify the gene. In case of clean deletion, you should not get any band if the gene is deleted.
Thank you all for your insights. I think I can't help it. the efficiency will definitely drop since it's my fourth gene to be deleted in that strain and I am just using the conventional FRT/FLP system
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