User:Sim Huay Ping/Notebook/CBE/08/150/2008/10/15

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Overview
Aim: To determine membrane depolarization activity of antimicrobial peptides on bacteria strains.

Equipments
Centrifuge Vortex UV Spectrometer Luminescence spectrometer

Materials
Bacterial inoculums (E. Coli, Pseudomonas Aeruginosa, Stahylococcus) 2ml Vials with lid Physiological saline buffer (PBS) Sterilized cuvettes Pipettes (0.1-10 ul, 10-100ul, 100-1000ul) HEPES buffer Membrane potential sensitive dye, DiSC3-5 AMPs prepared in vials (xB5, xB8, xB9) Gramicidin

Procedure
Note: Manipulating and transfers dealing with bacterial agents are to be carried out in the biological safety cabinet to prevent contaminations.

1. Obtain 2ml of bacteria from the tube of incubated, well-growing bacteria culture in broth, and transfer into a vial.

2. Bring the vial of bacteria inoculum to centrifuge at 14x1000rpm for 5 minutes. Throw away the supernatant.

3. Add 2ml of PBS into the vial and vortex it for 3 minutes. Bring to centrifuge again. Do this step of washing twice and lastly leave 2ml of PBS in the vial.

4. Transfer 2ml of HEPES and about 70 ul of bacteria in PBS into a cuvette. (This may vary according to the intensity of bacteria in broth). Measure the absorbance of the mixture using the UV spectrometer, using pure HEPES in another cuvette as reference for zero- ing the UV spectrometer. An absorbance reading of A600=0.05 is to be obtained as a standard.

5. Place the prepared cuvette of bacterial solution into the luminescence spectrometer. Settings for the luminescence spectrometer: Status tab – check that the instrument status is active. Sensitivity tab – Auto-range is set to 60% of the full range, and 665 volts for the detector. Monochrometers – Click on radio button for both excitation and emission. For excitation, set the wavelength to 622nm, step size at 1.0nm, bandpass at 4nm. For emission, Set the wavelength to 670nm, step size at 1.0nm, bandpass at 4nm. Application tab – Set the time trace to 1500 seconds.

6. Click on the green start button.

7. At 130 seconds, pause the run and add in 10ul of DiSC3-5 into the cuvette. Ensure that the lid of the spectrometer is closed completely. Resume the run immediately after addition of the dye and closing of the lid in order to capture the quick rise and fall peak in fluorescene reading.

8. At 500 seconds, pause the run again and add in 4ul of the AMP. Click on resume after replacing the lid.

9. At 1000 seconds, pause the run again and add in 40ul of Gramicidin. Gramicidin is used to observe if the AMPs have completely degrade the bacterial membrane previously. If there is further rise in fluorescence after addition of Gramicidin, it indicates that the AMPs have not completely degrade the bacterial membrane. Gramicidin acts as a positive control and has been used in excess.

10. Repeat the runs for each of the 3 AMPs on newly prepared cuvettes of bacteria.

Reference
Ka Hyon Park, Yoonkyung Park, II-Seon Park, Kyung-Soo Hahm and Song Yub Shin. Bacterial selectivity and plausible mode of antibacterial action of designed Pro-rich short model antimicrobial peptides. J. Pept. Sci. 2008; 14:876-882