I create a mutant deleting a GOI and I complemented it using a vector carrying the removed GOI. I checked my strains by PCR using two pairs of primer, a pair for the removed gene and a second pair with which I should be able to amplify flanking regions of the gene.
I obtained two bands in the WT (GOI, flanking region+GOI), one band in the mutant (Flanking regions noGOI) and two bands in the complemented mutant (GOI, flanking regions noGOI).
Then I saved mutant and comp. mutant in glycerol stock -80 and I used it in my assay.
The function I'm studying was eliminated in the mutant but not restored in the complemented strain so I checked again the glycerol stock of the complemented strain by PCR and I found that there were no GOI anymore
I repeated the complementation from the beginning, triparental mating, antibiotic selection and PCR, I saved the candidate strain and I used it in my assay but again, the function due to the GOI was not restored and the PCR showed no GOI band.
I would suppose my mutant lose the vector very quickly but I always use it with specific antibiotic in order to maintain the vector inside. There is a 4 hours step in my assay in which I need to growth it in media with no antibiotic, could be that the problem?
Do you have any suggestion?
A couple of stupid questions: Does the recipient strain already contain a plasmid? Is the Ori compatible with your recipient strain? Is your construct possibly producing a toxic protein?
There are several reasons a transformant can survive without a vector containing an antitbiotic resistence or reproducing it! Depending on the resistence, the protein expressed can exist for quite a while in the cell or on the membrane, further the media in which its growing can influence this as well.
One of my favorites is the phosphate concentration and kanamycin.
In a nut shell, your recipient doesn't like the vector/plasmid for some reason! And it doesn't need it to grow/survive in this 4-hour window!
First of all thanks for your answers guys!
I made sure my recipient strain doesn't contain the vector as well as the resistance carried by it before using it and I already use the same vector to complement a different mutant in the same recipient strain. Since everything was ok I would discard the idea of an incompatible Ori. Furthermore, I've checked the mutant and the complemented strain using PCR and combination of primers that should allow me to have a good idea of what there is inside my strain, in particular, if the gene is still in the cell/membrane I would have got amplification og the gene in the mutant as well.
The vector is carrying Km resistance and is a low copy number ones.
I though that since is a low copy number and my protein has a high native level of expression maybe is not enough expressed on the cell but still, I should recover at least a minimum level of function due to the low expression.
As I said before, a repeated the complementation twice getting the same results and I have no idea how to continue.
Any further opinion would be super appreciated.
What is your phosphate concentration in the media, minimum media?
Kanamycin and the phosphate concetration as I stated are not nice partners, if your phosphate concentration is around 50 mM?(not sure anymore) or above your will have problems retaining a plasmid, where the product may not be toxic, but is otherwise not well liked (recombinant plasmids are resource hogs).
You want selection media with a low phosphate concentration, might want to change to a complex media(likely not possible), or lower the phosphate concentration in your minimum media if this is the problem.
No, I'm using Lysogeny Broth media made by 10g tryptone, 5g yeast extract and 10g NaCl/ Not sure about the phosphate concentration.
Actually I tried before to perform my essay in M9 minimum media with no difference in the results (preliminary experiment) so I could repeat the experiment replacing LB with M9 during the 4h growing step/
Actually the phosphate concentration won't be a problem in complex media, or shouldn't be. You could try a minimum medium anyway, just take care of the phosphate concentration M9 has too high a P concentration, you would need to half it, and You could increase Kanamycin concentration. Are you centrifuging the culture and washing with something? Before placing in non-selective media.
You could also measure the growth curve during selection and afterward, which may give you some insight into how your plasmid is affecting the bacteria. If the growth curve is depressed (slow growth) during selection and perhaps lags afterward and is then exponential this would be a sign of negative selection.
One tip I have, would be to perhaps use an inducible promoter like 'lac' or "ara" with your construct if possible, if you still lose the vector then, well I'm stumped.
Further, you could isolate the plasmid from your test strain. You may be getting some kind of recombination eliminating the GOI. I don't know what the genomic background of your test strain looks like.
And as a last note Perhaps PCR for your GOI during different time points in the culture.
One last question for you to answer yourself, I know its stupid, but did you clone the entire promoter region and what does the promotion/supression system look like on your plasmid? Is your GOI itself involved in regulation of systems that could cause negative selection at the wrong gene dose or transcription level? Maybe a single copy plasmid would help?
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