Assessing the Antimicrobial Activity of Nisin Variants Using an Agar Well Diffusion Assay

Take a Lactococcus lactis culture expressing the membrane-bound peptidase NisP.

Mix the culture with a nutrient-rich medium.

Add molten agar, mix, and pour into a plate to evenly distribute L. lactis in the agar.

Once solidified, punch wells into the agar.

Next, take recombinant E. coli producing wild-type or modified precursor variants of the antimicrobial peptide nisin.

Centrifuge to pellet the bacteria, discard the supernatants, and resuspend the pellets in buffer.

Sonicate to lyse the bacteria, centrifuge, and transfer the supernatants to fresh tubes.

Normalize the lysates to equal optical density using buffer to ensure consistent peptide levels.

Pipette each sample into individual agar wells and incubate the samples.

During incubation, the Nisin precursor peptides diffuse into the agar and are cleaved by NisP, generating mature nisin.

Mature nisin inhibits L. lactis growth, forming clear halos around the well.

Compare halo sizes to evaluate the antimicrobial activity of nisin variants.

Add one milliliter of the Lactococcus lactis pre-culture to 25 milliliters of 2X M17 broth containing 10 micrograms per milliliter chloramphenicol and two percent glucose. Then, add 25 milliliters of lukewarm molten agar, mix, and pour the solution into a large petri dish. Keeping sterile conditions, dry the plate for 10 to 15 minutes.

Then, sterilize the ends of a glass Pasteur pipette by flame. After the pipette has cooled down, use its wide end to create holes in the solidified agar. Label the bottom of the petri dish for each sample next to the corresponding hole position.

For the activity assay, take the one milliliter samples of the E.coli production cultures and centrifuge them for three minutes at 7,000 times g. Aspirate the remaining medium and resuspend the cell pellet in 500 microliters of sodium phosphate buffer by pipetting.

Next, sonicate the sample on ice by submerging the tip of the sonicator probe into the cell suspension. Start the sonication. Then, centrifuge the cell lysate for 10 minutes at 13,000 times g to pellet cell debris.

Transfer the supernatant to a new reaction tube on ice. Dilute and normalize the supernatants to one milliliter of an OD₆₀₀ equaling 0.6, relative to the harvested cell density, with sodium phosphate buffer and mix. To test the antimicrobial activity, add 40 microliters of the normalized sample into the designated hole of the indicator agar.

Wait until all the sample is diffused into the agar. Incubate the plate overnight at 30 degrees Celsius. The next day, take pictures of the agar plate using a flat bed scanner or digital camera.

Growth inhibition halo size can be measured by hand or using software.