Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

We study multi-drug resistance efflux systems in gram-negative bacteria. We are interested in learning about the natural functions of these pumps and how these pumps can be used as a potential drug target. Expression of multi-drug resistance efflux systems is tightly regulated, making it difficult to study their functions.

Thus, an expression system that bypasses the regulatory system can be quite useful in the characterization of these pumps. This protocol allows for the expression of genes at levels usually seen in natural settings. Thus, the activity of the gene products mimics what is observed in nature.

To begin, place all sterile 1.5 milliliter microfuge tubes and sterile electroporation cuvettes on ice. Then place the bottle of sterile water that was stored at four degrees Celsius on ice. Transfer 1.5 milliliters of the overnight bacterial culture into one of the 1.5 milliliter microfuge tubes and centrifuge at 13, 000 G for two minutes to pellet the cells.

Using a one milliliter pipette, remove all the supernatant without disturbing the cell pellet. Add another 1.5 milliliters of bacterial culture into the same microfuge tube. Centrifuge at 13, 000 G for two minutes and remove the supernatant.

Add one milliliter of ice cold sterile water to the cell pellet and resuspend with gentle pipetting until the pellet no longer sits at the bottom of the microfuge tube. Centrifuge the resuspended cells at 13, 000 G for two minutes, and carefully remove the supernatant using a one milliliter pipette. Gently resuspend the final cell pellet in 200 microliters of ice cold sterile water, and transfer 100 microliters of the final cell suspension into the second ice cold 1.5 milliliter microfuge tube.

Prewarm a one milliliter of LB broth and one LB plus gentamicin auger plate for each sample and control in a static incubator set to 37 degrees Celsius. In a combined volume of five microliters or less, add 100 to 200 nanograms each of the pTNS2 helper plasmid and the pUC18T-mini-Tn7 tlac gentamicin, AdeIJK insertion plasmid to an aliquot of electrocompetent cells. Mix with gentle fingertip tapping to ensure complete mixing of the plasmids with the electrocompetent cells without introducing bubbles.

Add an equivalent volume of sterile distilled water into the negative control cell aliquot and mix gently as shown before. Incubate the samples on ice for 20 minutes. Transfer the entire cell sample into an ice cold electroporation cuvette, and then place the cuvette back on ice.

To electroporate the cell sample, turn on the electroporator and set it to two kilovolts. Wipe the surface of the cuvette with a soft tissue to remove any adhering ice or moisture. Insert the cuvette into the electroporator and deliver the electric shock.

Immediately add 0.9 milliliters of prewarmed LB broth to the cells in the cuvette, and gently pipette up and down to mix the cells with the media. Transfer the entire cell suspension into a new 1.5 milliliter microfuge tube at room temperature. Then check the time constant value on the electroporator.

For best results, this value should be between four and six. Incubate the electroporated samples at 37 degrees Celsius for one hour at 250 RPM to allow for cell recovery. After an hour, spread 100 microliters of each sample onto a prewarmed LB plus gentamicin auger plate using an inoculation spreader.

Incubate the plates at 37 degrees Celsius for 16 to 18 hours. Check the electroporation plates. Using sterile toothpicks, pick up to 10 single colonies from the LB plus gentamicin auger plates and patch them onto a fresh LB plus gentamicin auger plate.

Incubate the plates at 37 degrees Celsius for 16 to 18 hours. Patching transformation plate colonies onto selective media preserves the transformed strain and provides starting material for colony PCR screening. The PCR product for the screening primers, ABglmS2 forward new and Tn7 reverse is 382 base pairs.

Negative controls for the reaction include wild-type A.baumannii ATCC 17978, AB 258, and no template.