I am trying to insert into PBR322 araBAD promoter with my gene of interest(it should be inducible). Could you please tell wether I could use DH5alpha strain as I was told it lacks arabinose stansporter? Also my insertion contain region(about 1000bp) that is complement to the region that already exist in the plasmid, could you also suggest whether it is possible to do such cloning and what strains should I use in order to avoid recombination.
It looks like DH5a isn't missing any of the arabinose genes, the person who told you that might be thinking of DH10B/Top 10, which (supposedly) can transport arabinose but not metabolize it so it can be used to induce expression without the concentration changing over time. Also, I know people who use the araBAD system in species that naturally don't have a dedicated transporter for L-arabinose, as long as you use a relatively high concentration, enough (usually) gets in for induction, maybe by promiscuous passage through other transporters.
As far as your insertion being something that's already on the plasmid, that's probably okay in most recA- cloning strains. I've maintained plasmids that have a ~700 bp tandem duplication separated by
It looks like DH5a isn't missing any of the arabinose genes, the person who told you that might be thinking of DH10B/Top 10, which (supposedly) can transport arabinose but not metabolize it so it can be used to induce expression without the concentration changing over time. Also, I know people who use the araBAD system in species that naturally don't have a dedicated transporter for L-arabinose, as long as you use a relatively high concentration, enough (usually) gets in for induction, maybe by promiscuous passage through other transporters.
As far as your insertion being something that's already on the plasmid, that's probably okay in most recA- cloning strains. I've maintained plasmids that have a ~700 bp tandem duplication separated by
I can confirm that Alexandra's answer is correct both regarding arabinose and recA.
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