I have knocked out a gene in my bacteria (deletion by suicide vector) and I can observe a phenotype for that mutation. Then I inserted the same gene in a vector and transformed in E.coli which I used to transfer plasmid on my mutant (classic triparental mating). I sequenced the vector carrying the knock out gene before doing the mating and the sequence looks perfect from the beginning to the end. Actually I have also added a TAG sequence (FLAG) in order to use it for following experiment but I don't think this is the problem.
After the mating I checked the presence of the vector on my mutant by PCR amplification of the gene and the flanking regions of the same in order to check the absence of the gene inside the genome also.
I got the bands I expected so I started to use mutant and complemented strain for my experiment but the last strain is not able to recover the WT phenotype.
I repeat triparental mating twice just in case I did something wrong but no complementation again.
I re-checked the sequencing and I'm sure is ok.
I used the same plasmid for a second mutant before and it was ok.
Actually my plasmid is a low copy and I know my gene is highly expressed but still, I don't even recover partially the function I knocked out.
I also check protein expression by SDS page and the result confirm all the previous observations.
Any suggestion??
Hi there,
You wrote that "gene is highly expressed", do you mean mRNA is high and protein level is high? If so I see 3 basic reasons to explain the result:
High level of expression is detrimental to protein activity,
protein is not properly folded and therefore inactive,
the presence of the tag interferes with protein biological activity.
You also wrote " I used the same plasmid for a second mutant before and it was ok". Do you mean you use it to complement the effect of a mutation of the same gene or you use the plasmid for the complementation of the defect of a different gene? If the former it means the system is OK to express the active protein, if the latter it only tells you the system is OK for an other gene...
Hi Dominique, thank you for the answer.
I meant my gene is highly expressed in the WT, mRNA and therefore protein level so maybe a low copy number plasmid is not enough to recover the function.
How my protein could not be properly folded? What problem could have, maybe because of the FLAG sequence?
The plasmid I'm using for this gene is the same I used before for the same bacteria but different mutation so you are definitely right when you say the system is ok just for that other gene. Do you suggest to try with a different vector? Or maybe same vector but removing FLAG sequence? I may try both I think...
Hi again, I would first check the presence of the protein in the complementation assay. Then if present the issue would be is it functional? The easiest for this purpose is to check the protein behaviour during cell fractionation (does it come in the soluble fraction or in the cell debris pellet?). More difficult is to run an activity assay with cell lysate...
Protein misfolding may come from the rate of synthesis (if too fast it does encourage aggregation). BTW what is the promoter controling the plasmidic expression (if too strong it might be the reason for misfolding/aggregation).
Removing FLAG is also an option (it might interfere with protein activity).
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