Hello!
I wanted to know if anyone has ever experienced anything like this. I have tried subcloning erythromycin resistance gene to pCL55-itet vector. I confirmed that subcloning was success. I have streaking cloning DH5-a which get erythromycin resistance gene on LB+50 mg/L erythromycin agar. After 18h on 37 ℃, I check the plate and found both cloning DH5-a harboring erythromycin resistance gene and non-cloning DH5-a survive in erythromycin contained agar plate. I think non-cloning DH5-a couldn't grow on agar plate since It hasn't erythromycin resistance gene. Please help me to why this results happening! For reference, pCL55-itet has ampicillin marker not to erythromycin.
What's the erythromycin resistance gene coming from? If it's coming from another plasmid you might just be transforming E. coli with some of the source plasmid in addition to the legitimate clones. Otherwise, I'd try remaking your erythromycin stock and maybe increase the concentration to 150 µg/mL for plasmid maintenance - I don't think E. coli is particularly sensitive to erythromycin in general and I believe 50 µg/mL is on the low side for efficient selection.
Common mistakes are forgetting to add the antibiotic to the agar or using a strain that already carries the selection marker.
However, looking at your concentration of erythromycin it looks like you're using far too little. I would recommend using something like 250 ug/ml
Thank you for your help! I tried incubating on 200 ug/ml agar plate and found only cloning strains growth. I got result that I want due to your advice. However, I still wonder why DH5-a which is free eryrhomycin resistance gene could grow on 100 ug/ml. Would it be possible explain reasen about DH5a growth on erythromycin medium to me??
My guesses would be the colony you selected on 50 µg/mL erythromycin randomly acquired one of the following mutations:
But those are just assumptions, I couldn't find a solid source on spontaneous erythromycin resistance mechanisms immediately, I don't think erythromycin resistance mechanisms in E. coli have been studied quite as extensively as streptomycin and some other antibiotics.
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