I want to screen a Pseudomonas aeruginosa mutant library carrying a lacZ transcriptional fusion for dysregulating mutations. I tried to do it on xgal plates but the accumulation of the blue color makes the screening difficult. I saw that people are using macconkey or endo agar media for two-hybrid beta-gal screening in E. coli for instance, which would possibly allow the identification of colony which are hydrolysing lactose (from the change of color of the medium near the colony due to the change in pH?) from those which don't. I wonder if it would be something to try even with P. aeruginosa (which would here only possess the lacZ gene, so no permease etc...) and if someone ever tried it ? Or if other beta-gal substrates could be used for this application ? thanks. Julian
You need to have the lacY permease for those other indicator mediums like EMB or MacConkey, because they rely on hydrolysis of the lactose sugar and detect the change in pH. Detection by Xgal does not require lacY. However there are also fluorescent detractors of beta-gal expression which you could consider, but that would require instrumentation that you may or may not have access to.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology