I'm expressing fimbriae in two parts in my E. coli. Vector 1 contains everything except the tip (FimH), and Vector 2 contains FimH. Vector 1 is a pCRII-TOPO plasmid with ColE1 (pUC) origin of replication. Currently, my Vector 2 has a p15a origin, but I need it to be a much higher copy number, preferably closer to 100, although I'll take anything better than p15a. Any suggestions? These vectors are being expressed in DH10B cells.
Alternatively-- is there an easy way to put my ideal origin of replication into an existing vector that transforms well, expresses the ideal bacterial resistance (not ampicillin), etc.?
To increase its expression, and increase plasmid yield. I know I can do this by adding spectinomycin when growing it up, but it would also be better to have a more even ratio between the two plasmids.
We are making a library based on vector 2, so it's ideally we would express it at higher levels.
If you maintain selection for both plasmids (e.g., amp for one and kan for the other), you should be able to keep both plasmids in the cells.
No question about plasmid yield, but the link between copy number and expression is not that straight. Why don't you fiddle with the translational initiation signals of both constructs to obtain the desired product ratios instead? You can use something like the RBS calculator for this (http://www.voigtlab.ucsf.edu/software/)
By the way, Ronald is right. If the selection markers are different you can force two plasmids to coexist even if sharing the same replicon. Amp would probably be a bad choice, though.
Changing the promoter instead of plasmid copy number may work if you need to increase expression levels. Generally, T7 promoter gives high expression levels and is usually used for protein production in E. coli. trc promoter may also work the same way.
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