pCDFduet-1 plasmid dialemma

[eja77]

Has anyone had problems with pCDFduet-1 uptake by E. coli cells?

I am currently trying in vain to do coecpression of two proteins using the pCDFduet-1plasmid (to use one protein to improve the solubility of another). This plasmid contains spectinomycin antibiotic resistance. I have grown E. coli transformed with the pCDFduet-1 plasmid on agar containing freshly prepared spectinomycin solution (either 50mg/ml or 100mg/ml) [1:1000; antibiotic solution:agar]. The cells grow fine, and due to the antibiotic resistance, theoretically, only cells which have taken up the plasmid should grow. However, when I carry out a digest or PCR, no evidence of the plasmid is seen on the 1% agarose gel I run afterwards. I always run a positive and a negative control on the gel. The positive control always displays evidence of a plasmid, yet the samples mimic the negative control.

If anyone has any suggestions to explain this riddle (i.e. cell growth despite no plasmid with antibiotic resistance / causes of either no plasmid uptake or plasmid uptake but no evidence on the gel). Or any other bright ideas for coexpression of two proteins that is likely to work, I’d be very grateful.

Many thanks

Emily

[JLeo]

I'm not so familiar with the pCDFduet-1 plasmid or spectinomycin, but if I'm not mistaken, spectinomycin is similar to streptomycin? Certain point mutations in the rpsL gene can confer resistance to streptomycin, and perhaps this is the case with spectinomycin as well.

Which strain are you using? Have you checked that is is sensitive to streptomycin? How many transformant colonies do you get? If you only get a few (<10), these could simply be spontaneous mutants of rpsL and your transformation failed for some reason. But you imply that you are getting lots of transformants, so maybe the strain is already resistant? You could also switch plasmids and use one of the other pETduet vectors. I've had good success with pETDuet-1 (ampicillin resistance).