Generation of a Fluorescent Reporter Strain in Pseudomonas aeruginosa

Begin with an electrocompetent Pseudomonas aeruginosa culture suspended in a low-conductivity sucrose buffer.

Add a plasmid encoding green fluorescent protein (GFP) under a cyclic di-GMP-responsive promoter and an ampicillin resistance marker.

Mix and transfer the suspension into a pre-chilled electroporation cuvette.

Wipe and insert the cuvette into an electroporator.

Apply electrical pulses to form transient pores in the bacterial membrane. This facilitates the plasmid's entry into the cytoplasm.

After electroporation, the membrane reseals, trapping the plasmid inside.

Remove the cuvette, add fresh medium, mix, and transfer the bacteria into a tube.

Incubate to allow the bacteria to express the ampicillin resistance gene and GFP.

Spread the culture onto a nutrient-rich agar plate supplemented with ampicillin and incubate. The bacteria containing the reporter plasmid grow and form colonies.

Observe the plate under a fluorescence microscope. Green fluorescence confirms the successful generation of the fluorescent reporter strain.

Add one microliter of a 0.2 microgram per microliter solution of plasmid encoding the CdrA promoter to the gene encoding the green fluorescent protein to 40 microli