This article describes a method for generating a fluorescent reporter strain of Pseudomonas aeruginosa using electroporation. The process involves the introduction of a plasmid encoding green fluorescent protein (GFP) and an ampicillin resistance marker into electrocompetent bacterial cells.
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 microliters of the prepared P. Aeruginosa electro-competent cells in a 1.5 milliliter micro-centrifuge tube that has been pre-chilled on ice.
Mix the suspension and transfer it into a pre-chilled 2 millimeter electrode gap electroporation cuvette. Remove moisture on the outside of the cuvette with tissue paper and place the cuvette into the sample chamber of the electroporator.
Pulse the solution with a voltage of 2.5 kilovolts, capacitance of 25 microfarad, and a resistance of 200 ohms. Remove the cuvette and add one milliliter of LB medium.
Then, transfer the cells to a sterile 1.5 milliliter microcentrifuge tube and incubate for two hours at 37 degrees Celsius with shaking at 200 rpm.
Following incubation, spread 10 microliter, 50 microliter, and 100 microliter aliquots of the culture onto sterile LB agar plates supplemented with ampicillin and incubate the plates at 37 degrees Celsius overnight. Confirm the GFP expression by examining the plate under a fluorescence microscope with a standard GFP channel.
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