The overall goal of this methodology is to incorporate non-canonical amino acids into ribosomally synthesized antimicrobial peptides. New denature peptide are produced, checked for the presence of the ncAA and tested for antimicrobial activity using agar diffusion assays. This method can help to answer some key questions in the field of antimicrobials or bacteriaceas such as the role of certain peptide positions in the mode of action or in resistance development.

Following the same principles of variety of froops such as lesser peptides or can be produced and detectivity can be tested against pathogens such as MRSA. Peptides new to nature can be produced for the development of antimicrobial agents to fight pathogens and to overcome the deficiencies of antimicrobial drugs. The main advantage of this technique is that the variety of non-canonical amino acids can be tested in parallel without requiring changes on the genetic level.

Consequently, it is relatively easy to produce several modified Peptides in parallel. To begin this procedure, prepare 25 milliliters of LB medium containing antibiotics and one percent glucose in a 100 milliliter Erlenmeyer flask. Inoculate it using a colony of freshly transformed bacterial cells, or cells from a frozen cell stock.

Incubate the cells overnight at 37 degrees celsius and 200 rpm. The next day, add 10 milliliters of overnight culture to one liter of sterile fresh medium. Mix and split the culture into two 500 milliliter cultures each in a two liter Erlenmeyer flask and incubate at 37 degrees celsius and 200 rpm for two hours After two hours, measure the OD 600 using sterile medium as the blank.

Continue incubation and measure the OD at regular intervals until the OD 600 equal 0.5. Subsequently, pull both bacterial cultures and centrifuge at four degrees celsius and 4, 500 x g for 15 minutes. Then, pour off and discard the supernatant.

Resuspend cell pellet in centrifuge bucket placed on ice with 20 milliliters of NMM19 containing antibiotics and one percent glucose. Transfer the cell suspension into a 50 milliliter tube and centrifuge at four degrees celsius and 4, 500 x g for 10 minutes. Next, resuspend the cell pellet in five milliliters of the same fresh medium on ice.

Transfer the cell suspension into a 500 milliliters of fresh medium and incubate at 30 degrees celsius and 200 rpm for one hour. After an hour, divide the culture in two equal parts in individual flasks, one for each ncAA or control respectively. To each culture, add the stock ncAA to a final concentration of one millimolar and to the wild type control, add L-proline stock to one millimolar.

Then, induce target gene expression using one millimolar IPTG. Incubate the cultures overnight at 28 degrees celsius and 200 rpm. The next day, measure the OD and take one milliliter of each culture for the activity assays.

Collect the remaining cells of each culture by repeatedly centrifuging them at four degrees celsius and 5, 000 x g for 20 minutes. Each time, remove and discard the supernatant until all cells have been collected. Store the cell pellets at minus 80 degrees celsius until peptide purification.

To prepare GM17-agar plates, first prepare a five milliliter overnight culture of the indicator strain from frozen glycerol stock in M17 broth with one percent glucose and five micrograms per milliliter chloramphenicol in a 25 milliliter Erlenmeyer flask. Incubate the 30 degrees celsius without shaking. The next day, measure the OD using GM17 medium as the blank Add the five milliliters of fresh medium in a new flask.

Inoculate to an OD 600 equaling 0.1 and continue incubation. After two hours, measure the OD in regular intervals until the value reaches 0.4 to 0.6. Then place and keep the pre culture on ice.

Make 1.5 percent agar by weighing out 4.5 grams of agar in a glass media bottle. Add 300 milliliters of double distilled water, then mix and autoclave. Prepare two x M17 broth in 300 milliliters of double distilled water and autoclave.

Add one milliliter of the Lactococcus lactis pre culture to 25 milliliters of two x 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 x 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 x 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 OD 600 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 samples is diffused into the agar. Incubate the plate overnight at 30 degree 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. For fluorescence microscopy, prepare 10 millimolar Nile red stock solution in DMSO. For the activity assay, culture the indicator strain to OD 600 equals one.

Centrifuge one milliliter of the culture for three minutes at four degrees celsius and 5, 000 x g. Afterwards, discard the supernatant and resuspend the sample in one milliliter of PBS. Then, centrifuge the sample and resuspend again.

Subsequently, add one microliter of Nile red stock solution and mix gently. For microscopic image acquisition, add 30 microliters of cell preparation onto a cover slide while exciting at 520 nanometers. Set the acquisition time to 0.2 seconds, kinetic series to 0.1 Hertz and series length to 200 images.

Then, add 0.3 to 1.5 microliters of cell lysate or purified peptide samples. Monitor and record the fluorescence emission at a wavelength equal to or larger to 560 nanometers. Store the image sequences as avi files and analyze the images with ImageJ.

This figure shows the growth inhibition of the bacterial indicator strain by recombinant antimicrobial peptides containing proline analogs. As part of the agar weld diffusion assay, inhibition halos become visible around the sample positions. This video shows time lapse fluorescence microscopy of the indicator strain without the addition of the antimicrobial peptides.

Overtime, bacterial cells are deposited on the cover slide since only buffer was added, the appearance of the cells does not change within the 20 minutes of observation. This fluorescence microscopy time lapse shows the effects of an antimicrobial peptide on cells of the indicator strain. Overtime, the bacterial cells appear aggregated and blurry after exposure to a nisin variant containing the non-canonical amino acid 4R hydroxyproline as highlighted by the arrow.

Marked by circles, light material diffuses from the cells into the buffer indicating that membrane fragments were mobilized. This figure, shows the effects after incubation of the indicator strain in the present of wild type nisin or a variants containing non-canonical amino acids. After exposure to the antimicrobial peptide, the bacteria cells aggregate as shown by the circles.

Indicating cell lysates, light material becomes visible around the cells as shown by the arrows. Large amount of distorted and aggregated cells are present after 20 minutes of incubation in contrasts to the negative control. Once mastered, this technique can be performed in four days.

This includes production of the ncAA modified peptides, and activity testing using agar weld diffusion assays and fluorescence microscopy. While attempting this procedure, it is important to remember to use fresh bacteria cultures and suitable growth media and to handle them carefully. For activity testing, always include appropriate positive and negative controls.

Following this procedure, other methods of peptide purification by affinity chromatography and or HPLC can be performed. Mass spectrometry is mandatory to prove the incorporation of the non-canonical amino acid. After it's development, this technique paved the way for researchers to explore newer antimicrobial peptides other antibacterial or microbial host can be used for production on different peptides and for testing of the antimicrobial activity.

After watching this video, you should have a good understanding of how to incorporate non-canonical amino acids into antimicrobial peptides.