Measurement Background

In an alkaline environment, the peptide bond structure in protein molecules can form complexes with Cu(2+), and reduce Cu(2+) to Cu(+). The BCA reagent can specifically bind to Cu(+), forming a stable colored complex, with the maximum absorbance value at 562nm. The intensity of the color of the complex is directly proportional to the protein concentration, allowing the determination of protein content based on the absorbance value.

Measurement Principle

[Materials]
- FBSDs samples
- Reagent A:Configured in 1L ddH2O：BCA(10 g) ,Na2CO3 (20 g) ,sodium tartrate (1.6 g) ,NaOH (4 g) ,NaHCO(9.5 g)，pH=11.25
- Reagent B：Dissolve 4 g Cu2SO4·5H2O in 100 mL ddH2O
- 1 mg/mL BSA
- 96-well microplate
- microplate reader

[Procedures]

1. Mix Reagent A and Reagent B in a ratio of 50:1 to prepare the BCA working solution.
2. Add 20 μL of 1 mg/mL BSA and 20 mM Tris-HCl to wells A1-G7, ensuring that the amount of BSA added to each well matches the desired concentration shown in Figure 1.
3. Add 10 μL of the protein sample to wells A2-D2, and add 10 μL of 20mM Tris-HCl.
4. Quickly add 200 μL of the BCA working solution to each well and incubate at 37℃ for 30 minutes.
5. Measure the absorbance at 595 nm using a spectrophotometer and perform linear regression using Excel, as shown in Figure 2. The protein concentrations for samples 2A-2D are determined to be 114.94 μg/mL, 133.42 μg/mL, 155.98 μg/mL, and 79.42 μg/mL.

Measurement Background

Minimum Inhibitory Concentration (MIC) refers to the lowest drug concentration that can inhibit the growth and reproduction of microorganisms. There are many methods for MIC testing, with three common methods being: the broth dilution method, agar dilution method, and E-test. We have chosen the broth dilution method for testing (this method cannot detect anaerobic bacteria).

Measurement Principle

[Materials]
- Antimicrobial peptides
- Microorganism to be tested
- MH medium
- 96-well microplate
- microplate reader

[Procedures]

1. Cultivation of Staphylococcus aureus
Streak Staphylococcus aureus on an LB agar plate, and after overnight incubation at 37°C, pick a single colony and inoculate it into 4 mL of MH liquid medium. Incubate at 37°C, 220 rpm for 16 hours for later use.

2. Enzymatic digestion and dilution of FBSDSs
We believe that the antibacterial activity of the fusion protein comes from the defensins domain, not the binding domain. Therefore, we use the molar concentration of the fusion protein for MIC evaluation. Our previous antibacterial experiment results (refer to the Engineering success page through the provided link) indicate that the defensein domain needs to be released by sumo protease to exhibit antibacterial activity. Therefore, a sumo protease digestion reaction needs to be performed before conducting the MIC assay. Sumo Protease ulp1 from Beyotime (Cat: P2312S) is used for this purpose, with 20 μg of protein in a 200 μL reaction system (including 20 μL of 10× buffer). If the fusion protein concentration is below 117.65 μg/mL, add 180 μL of fusion protein directly for the reaction. After sumo digestion, conduct protein electrophoresis to confirm complete cleavage of the fusion protein.
The protein concentration after digestion (μM) = Fusion protein concentration before digestion (μg/mL) * [volume of fusion protein added (μL) / 200 μL] / molecular weight of fusion protein * 10^3. The molar concentrations of CBM3-sumo↓HNP1, CBM3-sumo↓HNP4, CBM3-sumo↓HD5, and CBM3-sumo↓HBD3 are 2.96 μM, 2.94 μM, 2.95 μM, and 2.00 μM, respectively.
Subsequently, perform 2-fold gradient dilution of the digested FBD↓S in MH medium. Add 100 μL of MH medium to a 96-well plate, then add 100 μL of the diluted FBD↓S to achieve the final concentrations shown in Figure 3 (using HNP1 and HNP4 as examples). Each group consists of 3 replicates; avoid bubble formation during pipetting, and if bubbles occur, use a sterile syringe needle to puncture them.

3. Determination of Growth Curve
Measure the OD600 value of pre-cultured Staphylococcus aureus and dilute it to 0.1 using MH medium. Transfer the diluted culture into sterile 8-well PCR tubes, then use a multichannel pipette to add 1 µL of Staphylococcus aureus to the 96-well plate containing FBD↓S (Control group without addition). Cover the plate, seal it with sealing film, and incubate at 37°C, 220 rpm for 12-24 hours. Stop measurements at the end of the logarithmic growth phase.
For the first measurement, readings were taken at 0 h, 0.5 h, 2 h, 4 h, 8 h, 12 h, 16 h, and 24 h to determine the absorbance at 600 nm. The measurement results are shown in Figure 4. Staphylococcus aureus enters the logarithmic growth phase between 8-12 hours; we selected 8 hours for determining the MIC value.

In our subsequent assays, we shortened the assay time to 12 h and the sampling interval to 1 h. More details could be found in our modelling page.

4. Determination of 8 h MIC value
Calculate the growth inhibition rate per well at the 8-hour time point as shown in Figure 5. The inhibition rate is calculated using the formula: [1-((absorbance value of test concentration - average absorbance value of Control group)/(absorbance value at 0nM - average absorbance value of Control group))] × 100%. The minimum concentration with an inhibition rate >50% is defined as MIC50, and >90% is defined as MIC90.

The 8-hour MIC50 values for CBM3-sumo-HNP1/HNP4/HD5/HBD3 against Staphylococcus aureus were determined to be 740nM, 368nM, 1475nM, and 1001nM, respectively. Additionally, the MIC90 values for CBM3-sumo-HNP4/HD5 were 735nM and 1475nM, respectively.

To determine a higher MIC99, the initial concentration needs to be increased, and the dilution gradient needs to be further reduced. For instance, in subsequent experiments, the MIC99 for CBM3-sumo-HNP1 was determined by adding 50 µL of 4×MH medium to the 96-well plate followed by the addition of 150 µL of CBM3-sumo↓HNP1 to achieve a higher concentration. The MIC and growth curve are shown in Figure 6, where the 8-hour MIC99 was found to be 2.37μM.