We are trying to find a method to characterize an efflux protein in S. aureus.
Usually done via change in antibiotic susceptibility as in "Multiple Novel Inhibitors..." in Antimicrobial Agents and Chemotherapy, Oct 1999 P. 2404-2408. Its a very simple method. Distinguishing between different MESs can be more challenging because you have to modify the genome.
Very interesting. Does your lab do this regularly? I couldn't help but notice you are at NC State not too far from where I am.
I'm using a similar assay currently to study where antimicrobial compounds are localizing in various species/strains of bacteria and in order to see the effects different efflux pumps are having on drug efficacy. We are chemists, not biochemists, so we aren't doing anything too fancy. I'm just making/testing inhibitors and interpreting the data. I doubt I'll do any cloning, since it is a bit outside of my skill set.
That is still very interesting work. I am a PharmD student at UNC and I have been working with Scott Singleton in the Division of Chemical Biology and Medicinal Chemistry looking at a gene that we believe is an efflux pump, and we have been trying to find ways to determine mechanistically what this gene product does. What family of efflux pumps are you looking at the most? How are you able to determine drug localization?
Nope, just a lowly grad-student. I've been looking at NorA (as in the paper I referenced) against S. aureus and a few other strains. I'm looking to expand to some other pumps specifically in Gram-negative strains. The localization aspect is specific to our work and wouldn't apply to antibiotics. As for your work... I would think developing a strain of staph. that had knock outs on all or most of its efflux pumps, then adding your gene on a highly expressed plasmid would convey substantial drug-resistance. You might be able to purchase or simply "borrow" a strain from another lab if you dig around. To begin to hypothesize what family of pumps it is, you could test out various inhibitors to remove the added drug-resistance.
you can use ethidium bromide and a fluorescence spec to measure accumulation and efflux. A general protocol is described here (Floyd, Jody L., et al. "LmrS is a multidrug efflux pump of the major facilitator superfamily from Staphylococcus aureus." Antimicrobial agents and chemotherapy 54.12 (2010): 5406-5412.).
Very interesting protocol. I have read that article? How noisy is this assay? Can you give me an idea about signal to noise ratio? Is the protocol user friendly?
Microbial drug resistance can be measured. A general protocol is described here (Gene cloning and characterization of MdeA, a novel multidrug efflux pump in Streptococcus mutans. J Microbiol Biotechnol. 2013 Mar;23(3):430-5 )
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