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Antibiotic marker expression in e.coli and geobacilli - (Nov/16/2005 )

I have recently cloned a resistance marker for a geobacilli species and as normal have perforemed all the cloning in E.coli. Although I do get transformants in the geobacilli strain I find it is to an efficiency only 1% of usual. When I take a rare transformant, extract the plasmid and put it back into my geobacilli strain the efficiency shoots up. However if I put it back into e.coli and then into the geo bacilli strain I still see low efficiencies. It appears any version of the plasmid that is shuttled through E.coli gives low efficiencies in the geobacillis strain. Anyone any ideas why this is?


The most likely thing is either presence of toxic methylation from the E. coli or the absence of methylation required to protect the DNA from a geobacillus restriction enzyme system.