Hello,
for my master's thesis, I have been working with phage display. One of my experiments involved electroporating a pUC19 vector containing the M13 full length gene 3, and a helper phage plasmid containing a super short version of this gene into TG1 E.coli cells. The pUC vector has no packaging signal whatsoever, yet when phage particles from this helper phage plasmid were allowed to infect empty TG1 cells, the antibiotic resistance of this pUC vector remained, suggesting that the plasmid, or part of it, is still present in the phage particle. Does anybody know what might have happened?
So two possibilities come to mind. The first is that what you think is pUC19 is actually something similar like pUC119 or pBS etc that do have an M13 packaging signal.
The second possibility is that you are just observing recombination between the pUC19 and the helper phage plasmid so that what is being packaged is a "dimer" of both plasmids. Remember that TG1 is recombination proficient.
You can easily test this by selecting for ApR transfectants and seeing if all of them also carry the drug marker from the helper. If 100% have both markers, then it is likely the second. If some only have ApR then it is likely the first.
You could also just grow package in a RecA- strain and see if the problem resolves.
Thank you very much for the answer, I'll look into those possibilities!
Robert Adolf Brinzer I don't think that is correct. The pUC18-19 series do not have a filamentous origin for packaging, but the pUC118 and pUC119 do.
Yes, I don't think I have the wrong type of plasmid either, as the plasmid has been used before without any problems by my supervisor in prior years.
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