I have constructed a plasmid containing a R6K origin and kanamycin resistance marker and Tetracycline resistance marker. I then transformed this plasmid into a F' strain with a tetR marker on the F' plasmid. However, I got very few colonies on both LB + Kan and LB + Tc plates. I constructed this plasmid in a pir+ strain and it went very well. Only I transformed this plasmid into this F' strain (pir+) gave me very few colonies.
What possible reason could explain this ? Could be the two Tetracycline resistance markers doing something to disrupt the plasmid? The genotype of this F' strain is
Δlac-169 rpoS(Am) robA1 creC510 hsdR514 ΔuidA(MluI):pir-116 endA(BT333) recA1 F'(lac+ pro+ ΔoriT:tet)
Thank you in advance !
There should not be any notable interference between F and R6K nor should it be a problem to have two Tet markers (except that you can't select for the plasmid one). However you state that you get few colonies on LB+Kan and also few colonies on LB+Tet. With the F' strain you should be unable to do a Tet selection, all the cells are Tet-R to begin with, so you should have a lawn there. If in fact you got very few colonies on Tet plates then you have a different problem somewhere, such as the strain (or the media).
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