Some of my backbone expression vector are clearly contaminated by this IS. I use DH5alpha or TOP10 for routine molecular biology and quite surprise to observe this type of insertions in construction deriving from them...
Many cloning strains have had their genomes sequenced and can be found in Genbank. You can easily check and maybe see if there are any standard strains that are free of this element.
Yes, it's present in both. Probably what's happening is there's a hot spot for IS186 insertion combined with a strong selective pressure for IS insertion in that area of the plasmid. Typically the selective pressure is to disrupt expression of a toxic gene. Occasionally it's the opposite where a gene that's necessary is poorly expressed (ie. an antibiotic resistance marker) and IS insertion increases the amount of mRNA.
One of the large gram positive bacterial genes I tried to clone in E. coli Top 10 only produced clones with IS1 insertions - all the insertions were independent events, but they all clustered in a specific region of the coding sequence. I had to switch to cloning in Lactococcus lactis.
Scarab Genomics has E. coli strains where literally all IS elements are deleted, which might be an option - but they're quite expensive, especially if you don't make your own competent cells.
If you can't switch strains, I would investigate what's up with the areas you're getting insertions.
Ho Okay, I've effectively seen it in the genome of DH5alpha but I did not find the genome of TOP10. Thanks a lot. In fact the insertion take place in the replication regulation area. May be it can influence the copy number to limit the toxicity of the cloned protein (a fluorescent protein). I will check on the website of Scarab Genomics to order a first batch of these clean E.coli ! Thanks again Alexandra !
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