The previous post discussed what I can call Problem area #1: the evidence that my plasmid prep results have been unreliable - that the absence of plasmid in the prep didn't mean that the cells I started with didn't contain plasmid. So now I need to go back through the other experiments, to check if my conclusions are still solid.
Problem area #2: Can the specR PCR fragment be ligated into a blunt-cut plasmid, when phosphorylation isn't needed? Answer: NO.
My first experiment said 'No', but it was flawed by using too high a ratio of plasmid to insert. So I repeated it using much more specR fragment than plasmid. This time the results were a cleaner 'No'. Religation of the cut plasmid gave 437 AmpR colonies for 510 µl transformation mix. Ligation of the same amount of plasmid in the presence of the specR fragment gave only 29 colonies from 150 µl of the transformation mix, and no SpecR colonies (from 150 µl) or AmpR SpecR colonies (from 150 µl). A positive control transformation using a plasmid that carries both AmpR and SpecR gave several hundred colonies of each from the same volumes.
So I conclude that the specR PCR fragment cannot be ligated, and that it also interferes with ligation of the blunt-end plasmid. One way to interpret this is that one end of the specR fragment is OK and the other is unligatable. I'm discouraged but not surprised by this result, because the specR PCR product always behaves oddly in agarose gels: a bit blurry before cleanup and worse after cleanup.
Solutions: (1) I could design new specR primers and try again. We might even have other specR primers for this cassette. (2) I could try amplifying with the original primers from a different template. The undergrad used a plasmid I think. I should have a plasmid with specR in a long-enough segment to encompass my primer sites.. (3) I could cut my existing specR PCR-product with NheI to generate sticky ends. This is what the undergrad had originally planned. I would need to design new primers for the inverse-PCR step that generates the rest of the desired plasmid. Or blunt-cut specR fragment out of a plasmid and ligate this to the inverse-PCR product.
Problem area #3: Are the kinase (phosphorylation) reactions working? Answer: YES.
I originally tried to test this by phosphorylating the inverse-PCR product and self-ligating it, transforming E. coli and selection for AmpR. I got no transformants, so either the kinase reaction failed or there was another problem. The ligation control, transformation control and plate-selection controls all worked fine. This was when I discovered I'd been using very old kinase, but repeating the experiment with new kinase gave the same result. Was the ATP stock bad? No, repeating the kinase reactions using ligase buffer (contains its own ATP) gave the same result.
A better test of the kinase function comes from the grad student, who has been using it to label chromosomal DNA with 32P from 32P-ATP. He's getting modestly successful incorporation, suggesting that this reaction is working OK.
Problem area #4: Is the inverse-PCR product's intact toxin gene toxic to E. coli? Answer: Weak No (no evidence that it is).
I tested this by making a different inverse-PCR product using the undergrad's old primers. These cut off the last 5 amino acids of the toxin gene. She was able to get successful ligation of this product to her specR PCR product, after kinasing a mixture of both fragments. I ligated this with my kinased SpecR fragment. This produced one AmpR SpecS colony and one AmpS SpecR colony. The negative control self-ligation of the inverse-PCR fragment alone (not kinased) gave a few AmpR colonies. If these do result from self-ligation (I didn't do a plasmid prep on them).
I had only kinased the specR fragment, because I didn't want the inverse-PCR fragment to be able to self-ligate. In retrospect, especially now that I know that the specR fragment is unligatable, I should have also kinased the inverse-PCR product as a better control to show that the kinase was working.
THE BIG QUESTION: SHOULD I KEEP TRYING?
This project shouldn't have been such a big deal. But it's the bottleneck in getting the toxin-antitoxin work finished and published.
One possible plan: Buy some NheI, cut the (blurry) specR PCR fragment and run it in a gel. Do I get a nice sharp fragment of the right size? If yes, design and order new inverse-PCR primers with NheI sites. (Why did the undergrad choose NheI? I have no idea?) Cut the new inverse-PCR product with NheI and ligate it with the specR fragment. Promers are cheap, so I oculd instead jsut design new specR and inverse-PCR primers with matching sites.
Another plan: I recently found a note I made at last summer's Gordon Conference, reminding me that a favourite colleague had offered to make this damned mutation for me. But I don't like to ask this of him...
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