We are having trouble cloning a human gene of unknown function into a plasmid that is supposedly replicating (not translating) in E.coli.
We have tested three E. coli strains (TOP10, DH5alpha, XL1blue) but do get only very few colonies that are very small in size. Sequencing plasmid minipreps from these colonies revealed that each plasmid has a deletion (individual deletions per mini) located in the 3' end of the gene ORF.
Our guess is that this gene is expressed at low levels and kills the cells.
Are there any tricks out there to overcome this problem? We cannot easily change the plasmid either.
Any comments would be highly appreciated, thank you!