Summary
As a phage-derived enzyme, endolysin is essentially a protein molecule that assists in the release of phage particles from the bacterial host by breaking down the peptidoglycan component of the bacterial cell wall, and can be expressed during phage replication, disrupting key chemical bonds in the peptidoglycan of the bacterial cell wall.
Principle
The lysis effect of endolysin on host bacteria can reflect its antimicrobial activity. After co-incubation of endolysin with host bacteria, the change of OD600 before and after the addition of endolysin was counted by turbidimetric assay, the number of viable bacteria was determined by bacterial counting method, and the antimicrobial activity was detected by observing the morphologic difference of host bacteria before and after the addition of endolysin under the light and lens microscope.
Appliance
Potential solutions for the treatment of antibiotic-resistant bacterial infections.
Operation method
Endolysin activity assay
Principle
The lysis effect of endolysin on host bacteria can reflect its antimicrobial activity. After co-incubation of endolysin with host bacteria, the change of OD600 before and after the addition of endolysin was counted by turbidimetric assay, the number of viable bacteria was determined by bacterial counting method, and the antimicrobial activity was detected by observing the morphologic difference of host bacteria before and after the addition of endolysin under the light and lens microscope.
Materials and Instruments
Instruments: Move 1, OD600 determination and analysis: Single colonies of host bacteria were picked and inoculated into LB medium overnight, and then inoculated into new LB medium at the ratio of 1:100 on the next day and incubated for 3 h until the OD value reached between 0.8~1.0. 1.5 mL of the bacterial solution was centrifuged at 16000 g for 2 min, washed twice with PBS and resuspended in 500 μL of Tris-HCl buffer (containing 1% TritonX-100), and injected into a 96-well plate with 180 μL in each well. 20 μL of purified endolysin protein with the final concentration of 2 mg/mL was added to the experimental group, and 20 μL of reaction buffer was added to the negative control group, which was detected by a multifunctional enzyme marker. The reaction buffer was added to the negative control group, which was detected by multifunctional enzyme labeling instrument. 2. Bacterial counting method: Pick a single colony of host bacteria and inoculate it into LB medium overnight, then inoculate it into new LB medium at the ratio of 1:100 the next day and incubate it for 3 h until the OD value reaches 0.8~1.0. Take 1.5 mL of the bacterial solution and centrifuge it at 16000 g for 2 min, wash it twice with PBS and resuspend it in 500 μL of Tris-HCl buffer (containing 1% TritonX-100), and take out 10 μL of the sample as the zero-moment negative control group, and then add the purified endolysin protein with the final concentration of 2 mg/mL into the original bacterial solution, incubate for 30 min at 37 ℃ and take out 10 μL of it as the experimental group, and take a 10-fold dilution of it as the experimental group, and then add the endolysin protein into the original bacterial solution. As the experimental group, the 10-fold dilution method was adopted to count the coated plates of each group, and the average value was repeated three times. 3、Light microscopy analysis: Pick a single colony of host bacteria and inoculate it into LB medium overnight, then inoculate it into new LB medium at the ratio of 1:100 the next day and incubate it for 3 h, until the OD value reaches 0.8~1.0. 1.5 mL of the bacterial solution was centrifuged at 16000 g for 2 min, washed twice with PBS and resuspended in 500 μL of Tris-HCl buffer (containing 1% TritonX-100), and 20 μL of purified endolysin at a final concentration of 2 mg/mL was added into the experimental group, while 50 μL of the samples of the negative control group was added into the reaction buffer, and the samples were stained with Aquamarine for observation under a light microscope. The samples were stained with Aquamarine and observed under light microscope. 4. Transmission electron microscopy analysis: Sample preparation was the same as the light microscope sample, 50 μL of sample from each group was dropped onto a copper grid with a film, after a few minutes of resting, the excess liquid was sucked up with filter paper, and the sample was negatively stained with drops of 3% potassium phosphotungstic acid, and then observed under the electron microscope after drying.
Centrifuge, multifunctional enzyme marker, light microscope
transmission electron microscope
Reagents:
Host bacteria (Kp3, CRKP, etc., can choose more than one)
LB medium, PBS buffer
Reaction buffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.4)
3% potassium phosphotungstate
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