JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:37 min • October 30th, 2025
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 lukewa
View the full transcript and gain access to thousands of scientific videos