Is there any alternative to x-gal white/blue screening for the monitoring of pseudomonas aeruginosa beta-galactosidase activity on agar plate?

Question

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. I wonder if it would be something to try even with pseudomonas (which would only possess the lacZ gene, so no permease etc...) and if someone here ever tried it ? Or if other beta-gal substrates could be used for this application ? thanks Julian

Answer 1

I think your problem will be even worse when you'd use MacConkey or Endo plates. You can see differences between different kinds of bacteria / expression of certain enzymes on those plates, but the coloring / decoloring is based on soluble compounds that easily diffuse.

Hydrolysis of xgal creates an insoluble product that's staying more or less at your colony. Of course it's not perfect and after a while you have a completely blue plate, but this would happen much faster with a soluble compound.

Try adjusting your conditions so you can look at the plate before it's completely blue. Things you could try: look earlier, plate them a little bit more dilute so colonies are further apart, use less xgal so your final coloring is less intense, look for some papers that use xgal for screening and steal their tricks.