I am setting up a competition assay where I'll be competing E coli strains against each other. Each strain is genetically marked with a fluorescent protein, e.g. CFP and YFP. Can anyone recommend a good plasmid/promoter choice for this? I was thinking something like pBAD or pET28 but someone told me they thought pBAD would result in unstable expression but I don't understand why.
I'm not sure what is the meaning of "stable" here, do you need
to maintain some level of expression throughout growth experiment?
If yes, I would clone your protein under non-inducible promoter
(you may substitute resistance marker on the plasmid with your protein,
and add another resistance marker elsewhere). Promoter of TEM-1
(common Amp resistance marker) was studied well, and you may tune
expression levels by mutations).
From limited personal experience, GFP had visible effect on the fitness
of the bacteria, meaning it is not ideal to track what is going on during
competition.
Amanda,
You can try pRSET A,B,C from invitrogen, they are good with special benefit of low size. Another choice on pET is perfect
I am guessing what you want is constitutive, low level expression (high level would be selected against in a long-term culture). I would recommend going for chromosomal insertion (rather than expression from a plasmid) and using the bla promoter, as recommended by Mike above.
Both of the plasmids have work well in my hands. With pET plasmids, expression might be leaky while pBAD systems are bit strict in control.
Remember to check the genotype of the E. coli strain while you use any of your plasmids, some strains might not be compatible that would yield bad expression profile. With pBAD you are limited to strains like TOP10(deleteion of ara) and pET systems require lambda lysogens.
And disregard of any plasmid and strain, every proteins behaves different and you need to fine tune your system after initial trials.
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