How hard could it be?
As part of a collaboration on the effects of very low concentrations of antibiotics on gene expression (topic for a future post), we need a mutant that is highly resistant to the antibiotic erythromycin (EryR). Erythromycin kills bacteria by blocking their ability to make proteins; it's fairly widely used and resistance mutations are common. These mutations have been studied in a number of different bacteria including H. influenzae.
Resistant strains usually have point mutations that change the large subunit of ribosomes (where protein synthesis happens); either in the genes for the ribosomal RNA (rRNA) or in the gene for either of two ribosomal proteins, called L4 and L22.
Erythromycin-resistant H. influenzae have been isolated from patients after treatment with the antibiotic. But 'clinical' strains (from sick people) are genetically very diverse, and we needed the resistance mutation to be in our standard lab strain KW20. So our first step was to try to isolate our own mutant. In principle this is easy - just plate KW20 on agar containing enough erythromycin to kill normal sensitive cells. But we (i.e. the student working on this) never got any EryR mutants.
Then we decided to try to transfer an EryR mutation from a clinical strain into our KW20 strain. We found a paper from a lab that had studied EryR strains, sequencing the rRNA and L4 and L22 genes to identify specific mutations. They found strains whose L4 and L22 mutations made them highly EryR. So we emailed them and they kindly sent us DNA from two strains (one L4 and one L22 mutant). We had them send DNA rather than bacteria because importation of H. influenzae from the US into Canada requires a government permit and we didn't want to bother with the bureaucracy.
Our plan was to use PCR to amplify the DNA containing the mutations and transform this DNA into KW20, selecting for EryR cells by plating on erythromycin agar. The PCR seemed to work fine, but the transformations never produced any EryR colonies. The cells could be transformed with other antibiotic-resistance genes, but because we didn't have any other EryR strains to use as positive controls we couldn't figure out why we got no EryR mutants.
So the student went back to reading papers, and discovered that the same lab that had sent us the DNA had, in an earlier paper, described doing just what we were trying to do - transform the EryR genes into KW20. And unlike us they were successful! So she contacted the lab again and this time we're going to have them send us the resistant strain, not just the DNA. We're waiting for the permit to arrive (note to self: go check the inconvenient fax machines tomorrow).
She's back, again, again, again!
14 hours ago in The Phytophactor