Recap of the last few posts: Starting with a plasmid containing the toxin-antitoxin operon of Actinobacillus pleuropneumoniae, I've been trying to create a derivative plasmid whose toxin gene is functional but whose antitoxin gene has been replaced by a specR cassette. This involves several steps: PCR of the specR cassette and inverse-PCR of the plasmid (to produce a fragment lacking the antitoxin gene), phosphorylation of the specR fragment with T4 kinase, ligation of the two fragments, transformation into E. coli, and selection for SpecR and AmpR cells. The PCR steps work but the rest fails to produce any resistant colonies
After the first attempt failed I introduced several controls: EcoRI-cut pUC18 as a ligation control, another SpecR AmpR plasmid as a transformation control, and the kinase-treated inverse-PCR fragment as a kinase control. The ligation and transformation controls worked, so I decided the kinase was at fault. This hypothesis was supported by finding that I had been using a long-expired stock of kinase rather than the one bought earlier this year.
But two more attempts using the new kinase have also failed. The first used the supplied kinase buffer and my stock of ATP, and the second used new ligation buffer (recommended by the supplier) which contains its own ATP. The second time I also preheated and rapid-chilled the substrates, which is recommended to help expose the blunt ends to the kinase. Both times I got no transformants from either the test or the kinase control, but got lots with my transformation control plasmid. (I didn't bother repeating the ligation control.)
I've been trying to think of what else could be going wrong with the kinase reaction, but it just occurred to me that maybe there's a completely different problem - maybe this toxin is toxic to E. coli.
Although the honours student had mentioned this concern when she handed this project over to me, I had discounted it because the H. influenzae homolog is not toxic in either H. influenzae or E. coli. But both my desired construct and the recircularized inverse-PCR fragment I'm using as a control are expected to express the toxin, possibly at high levels. So maybe my reactions are all working, but the plasmid they produce is not tolerated by E. coli.
How to test this? Directly testing for lethality is tricky. But I can do a different kinase control using a different inverse-PCR fragment, one that won't express the toxin. If the problem is the toxin, this should give AmpR transformants. I can also use the same fragment with the spec cassette, and I should now get SpecR AmpR transformants. I have all the honours student's primers and her CR conditions so this should be straightforward.
End of summer &
12 hours ago in The Phytophactor