Team:SMS Shenzhen/Demonstrate

Result

We demonstrate that our project is effective in LL-37 and the motor protein yebf through the experiments below.

Antimicrobial Effect of LL-37:

To test the wide anti-bacterial function of LL-37 expressed by E.coli, we selected three commonly found representative bacteria including both Gram-positive and Gram-negative Bacteria: Staphylococcus aureus, Bacillus subtilis, and the not genetically modified Escherichia coli, using the LB culture for cultivating the bacteria.

First, we spread the liquid bacteria on the LB solid in petri dish.

After the film of liquid bacteria is completely dry on the LB solid culture, we separate the petri dish into four parts as the diagram below shows.

In this experiment, water is the negative control and antibiotic carbenicillin as the positive control. On each section, we add a drop of 10μL of the corresponding liquid. After all the liquid is dry, the petri dishes are placed in 37℃ incubators for cultivation.

We set up comparisons as well, one section is added with induced yebf-LL-37 as the experiment group; another one is added with yebf-LL-37 that had not been induced as the control group.

Figure 1: Antimicrobial effect of yebf-LL-37 against Bacillus subtilis

Figure 2: Antimicrobial effect of yebf-LL-37 against Staphylococcus aureus

Figure 3: Antimicrobial effect of yebf-LL-37 against E.coli

The results matched our expectation and proved our antimicrobial concept. For all three types of bacteria, yebf-LL-37 that had not been induced did not produce an antibacterial effect while ampicillin is effective.

For the part added induced yebf-LL-37, we can see a clear circle in the middle of each section with induced E.coli, which mean that LL-37 effectively inhibited bacterial growth in B.subtilis and S.aureus. The effect of it in E.coli is not so obvious, which may due to that the chassis is E.coli.

From this experiments, we proved that the antimicrobial peptide LL-37 is effective against both Gram positive and Gram-negative bacteria.

Figure 4: Effect of yebf for LL-37

Effect of motor protein yebf：

[To clarify the three samples on the figure: The results of the previous two lanes, the tenth and the eighth, are sufficient to conclude the effect of yebf. The third sample is just for our team members to experience using liquid nitrogen. ]

Lane 8:

We first induced the bacteria expressing yebf-LL-37 over night under 28 Celsius. Then we centrifuged 1.5 mL of the product and obtained the supernatant as our sample. Therefore, only the protein outside the E.coli cell membrane in the liquid culture can be detected.

Lane 9

We first induced the bacteria expressing yebf-LL-37 over night under 28 Celsius. Then we centrifuged 1.5 mL of the product and discarded the supernatant, leaving the bacteria deposit. After that, we added the protein extraction reagent to the deposit to decompose the bacteria, thus to release the proteins inside the membrane, if there were any. Then we put the sample on ice for 20 minutes for the reagent to completely decompose E.coli. After that, we centrifuged the liquid, obtained the supernatant as the final testing sample and discarded the bacteria deposit. In this case, only the protein inside the E.coli cell membrane can be detected.

Lane 10

We first induced the bacteria expressing yebf-LL-37 over night under 28 Celsius. Then we obtained 50μL of the product and used liquid nitrogen to repeatedly thaw and refreeze the bacteria’s cell walls. At last we centrifuged the product and obtained the supernatant as our testing sample.

The result is obtained as followings:

Since LL-37 is fused with the motor protein yebf, the protein should be already transported into the culture rather than inside the bacteria. Seen from the figure, the result is consistent to the theoretical result. Since the motor protein yebf should have already taken LL-37 outside the cell membrane into the liquid culture, there should not be any test of LL-37 in this lane. Seen from the figure, the result is consistent to the theoretical result.

The protein is not detected inside the cell membrane (Sample 2), while it is detected outside the membrane in the culture (Sample 3). Comparing these two results, we proved that yebf is effective in carrying LL-37 out of the E.coli cell membrane.

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