Hi
I have experiment with Klebsiella oxytoca for gene integration by homologous recombination.
I have designed the linear DNA cassette with target gene and Cm. as selection marker. And The homology sequences were 300bp in front and 60 bp at other side.
The transformation was done by electroporation, and the universal integration procedure was used.
In plate incubation, I can get about 100 colonies in plate with selection antibiotics.
However I can not get any success in gene integration at a specific site.
(I can check it by colony pcr where the band will be detected only when the success in gene integration. but I can not any band in colony pcr)
If you had similar problem and solve it, plz help me.
Thank you.
'm assuming you ran a negative control for your transformation and didn't see any colonies. If you didn't, running that control is your first step. Otherwise, here are three possibilities to get to the bottom of what's going on:
1. Your PCR may not be working. It sounds like you only see a band when the integration is successful, so you may not have a positive control with which to verify that the PCR is actually working. You may need to vary your PCR conditions to get this to work- try using a single colony that grew on selection, and trying a range of annealing temperatures and DMSO concentrations (0, 3 and 5%.) You may also need to increase your extension time. When working with genomic DNA, polymerases move more slowly than with plasmids or PCR products because they must knock off proteins that are bound to the DNA.
2. Design a different PCR strategy. Strategies that use presence or absence of a band can be difficult to interpret when things go wrong. Instead, design primers just outside the insertion site to look for a small vs. large band. This should at least clarify whether the insertion is present or absent, regardless of whether your previous PCR design worked.
3. Check for homology of your cassette to anywhere else in the genome. I had a similar problem to the one you're describing when I tried to integrate an auxotrophic marker as a selective marker with my gene of interest. Although I used 100bp homology arms on either side of my cassette, my integration happened 100% of the time at the native gene rather than at my target site. This is because the homology with the native gene (which was mutated to be inactive, but still present) was much more extensive than the homology with the target site. You can use BLAST to check your cassette against the entire klebsiella genome and look for other regions of similarity where your cassette might be preferentially integrating. If this is the case, you'll need to redesign your cloning strategy.
Dear Sarah Stainbrook
Thank you for your kind response.
I agree that it's hard to interpret the results of colony pcr in my case, so I decide to take your advice. (It's now under experiment)
However, If all the colonies show negative results (no integration), then what should I can do??? plz tell me your experience of overcoming the problem.
Of course, I have checked several times about my DNA cassette and confirmed that there is no homology region except my target site in the genome.
(Some homology sequences detected in BLAST show below 20bp)
Thank you
In addition, I can get ban as positive control in pcr. However, interestingly I can not get any band even when pcr was conducted by two primers which were expected to present a band no matter what ( wild or integrated one).
What are you using as your positive control for the PCR?
Is there any possibility that your gene that you're integrating could be toxic? For example, does it produce a membrane protein that, if overexpressed, could disrupt the integrity of the membrane? Does it disrupt an essential gene, or has this site been used before in Klebsiella? Is it under too strong a promoter, such that it might provide too much of a metabolic burden on the cell? Could the protein be misfolding and overwhelming the cell?
Occasionally, weird genomic rearrangements can occur. Even if this was a rare event, depending on the time you recover after transformation but before plating, that one colony may have reproduced and made many offspring. You may want to order a single degenerate primer that can bind anywhere in the genome, and use that with one other primer in a touchdown PCR to amplify a band (any band is fine) for sequencing with your other primer. This will tell you what's going on.
A second thing to try is sequencing your target site in the parent strain, just to make sure that your homology region hasn't been mutated in the parent strain.
If you're still having trouble, it might be worthwhile to check whether you have a contaminant. You can use 16S rRNA sequencing to verify the species of the organism you're working on.
Sometimes, weird things just happen. It might solve your problem to go back to a frozen stock of the parent strain, streak it out fresh, and retry the transformation with fresh reagents and a sequence-verified insertion cassette.
The final option is to redesign your cloning strategy- choose a different location in the genome, use longer homology arms, perhaps change the promoter strength, etc.
Good luck!
http://bitesizebio.com/18992/a-primer-for-designing-degenerate-primers/
Thank you
I will share my experience later if I success in my experiment .
- Badges
- Science topic