I need a plasmid to complement a phenotype in a clinical Providencia rettgeri strain. I have found a few papers of Providencia stuartii or Proteus, where they use pGEM-T or pBC KS(–), to express the protein by mean of lac promoter in these specie. In those cases they use modified strains. However, I'm not sure if this lac promoter is going to work in my Providencia rettgeri... Does anyone know a plasmid option to complement a phenotype in this kind of bacteria or something related?
Thanks!!
I haven't done much work with Providencia rettgeri but I have done quite a lot with Providencia stuartii which is one of its closest relatives. I think the lac promoter will work but will be constitutive, not inducible, unless the plasmid you're using encodes for lacI. I know the Em7 promoter works in P. stuartii, though again, is constitutive. The lambda PL promoter with the cl857 mutant inducible by heat shock works in P. stuartii. As does the tet promoter which can be induced with anhydrotetracycline, which induces the promoter without killing tet sensitive bacteria.
Be cautious if you use an ampR plasmid - Providencia species are all somewhat ampicillin resistant and you will get a lot of background colonies if you try standard concentrations. Typically when selecting for a plasmid in P. stuartii that's ampicillin resistant I use carbenicillin instead of amp and use 200 ug/mL for initial selection - after selection you can lower the concentration down to 100 for routine maintenance. I'd also recommend screening for the plasmid by PCR to make sure it's actually there and you don't have a spontaneous mutant - spontaneous antibiotic resistance seems to be extremely common in general with this genus.
By the way if you run into any issues in the future, feel free to message me - with P. stuartii I've run into a ridiculous amount of issues (exacerbated by working with an MDR strain that's not very cloning friendly) and have figured out a lot of methods/workarounds.
Hey Alexandra!! Thank you so much!! Your answer is absolutely useful to me!!! I'm working with a clinical MDR Providencia rettgeri too!! This strain is resitant to all b-latams and aminoglycosides, so, we think to modified the plasmid.
Glad I could help. Do you know if it's resistant to Hygromycin B? It's technically an aminoglycoside but it can't be used clinically due to its toxicity to mammalian cells so specific resistance in clinical strains is rare. That's what I typically use for my strain of P. stuartii, though it requires 150-200 µg/mL for effective selection without background colonies.
Thanks Alexandra, I'm reviewing your Proteus paper. You have a lot of amazing information there!! I think that your Hygromycin B option is a really good idea for us, however we do not have a template for this resistance cassette... we're going to test the resistant to this antibiotic in our P. rettgeri strain. Again thanks a lot!!! I think I'm going to bother you with some silly questions later, sorry!!!
- Badges
- Science topic