ColEI incompatibility - (Jun/03/2010 )
Hi, I have a plasmid ( say p1) which has incompatibility with ColE1 plasmids. My question is, if i remove a gene from p1; say gene 1, and introduce into a plasmid ( say P2) which has colEI replication origin, will gene1 be expressed??
Also, can any one give me the molecular basis of how to get the best expression of the gene, like what factors we need to consider, where we should put the gene etc..
Also, say I need to replace gene A with gene B whose promoter is 2000bp away from the gene. Should I put it where Gene B is present or beside the promoter of the gene B?
Thanks for any advice.
i'm afraid i can't really follow your descriptions ...if you want help try to explain your problem in more detail.
Concerning high-level expression:
A promoter 2000bp upstream of a gene would not initiate expression of your gene in E. coli. Normally, promoters in E. coli are found within a region of 100 bp upstream of a ribosome binding site. A detailed review on the design of a prokaryotic expression unit can be found here.