Chromosomal Gene Deletion - Gene Deletion by Double Crossing Over in Pseudomonas (Feb/28/2008 )
i'm trying to delete (not knock out!) a 24kb fragment in Pseudomonas syringae.
Until now, i prepared a suicide vector (pME3087) to insert a Tetracycline resistance. With this i select for a first crossing over event.
In the vector are two flanking fragments of 700bp, on each side of the 24kb fragment one. This fragments have been combined by
overlap extension pcr.
The first crossing over event worked fine, i could confirm by pcr and tetracyclin resistance selection very good.
Now for the second crossing over event i try to enrich cultures with colonies which "kicked out" the insertion.
My protocol is like this:
- Adding Tet (20ug/ml) to hold/stop growing sensitive ones (those which done a 2nd crossing over).
- Adding Carbenicillin (2mg/ml) to kill Tet-Resistants because they're growing further on.
- Wash with LB without Antibiotic and grow O/N
After four cycles i do replica plates with around 100 single colonies.
Unfortunately i could not find any colonies which showed the second crossing over event.
Any suggestions? Please help me!
If i understand correctly what you are trying to do, you are basically doing an enrichment using carbenicillin. However, this logically depends on having some correct mutants to enrich. Also, as i remember, enrichment is not actually that great a proceedure (nowhere near perfect). However, having made unmarked deletions in Paracoccus, I have a suggestion...
After you have your integration strain after the first crossover, how many times do you restreak the strain before the enrichment? I found that the more times you restreak (i generally do 3 restreaks), the more correct mutants you have.
As I see it, you need to do restreaks before you enrich, otherwise you won't have double crossovers to actually enrich?
Hope this is of some help.
The other option is to try to get hold of a suicide vector which also integrates a lethal gene so that all of your single crossovers can be eliminated during the screening process...
You need a good conditional negative selection, against the insertion. The sacB gene is one possibility, but people have trouble with it. Another is the pKSS plasmid (Kast) which is a point mutation in pheS, the phenylalanine tRNA charging enzyme. It's dominant negative, conveying lethal sensitivity to the presence of p-chlorophenylalanine in the medium.
thx for your suggestions. normally i streak out the 1st crossover candidates only one time, then i enrich with single colonies.
you really think that repeated streaking out would enlarge the positive colonies for a second crossing over? any idea why? or is this just
anyway, i'll try this and will report here after this my succes (hopefully:))
the idea with the lethal genes is good as well, but then i need to clone newly. and therefore i prefer the first method.
The main reason I can think that the repeated restreaking helps (apart from the knowledge that in my case it definitely does) is that you get more rounds of cell division and subsequently more chance of a second crossing over. No hard evidence for this i'm afraid!
If i'm understanding you right, i could then select on positive clones which undergone a 2nd crossing over (cause of my bad english knowledges i had some problems to understand Kasts Paper correctly)? Means, i could prepare media which contain p-cl-phe and only those are surviving which kicked out the pheS? Obviously this would be a really good strategy... if the first strategy with replica plate fails i'll try this!
Exactly. Warning -- the pH of the p-chlorophenylalanine medium is important. Adjust it after autoclaving to 7.5 or 8.0. The p-chlorophenylalanine will not dissolve prior to autoclaving it.