Team:Beijing 4ELEVEN/Part Collection
Part collection:
BBa_K3089006, BBa_K3089007, BBa_K3089008, BBa_K3089009, BBa_K3089010.
Through article research, we collected 5 Antimicrobial peptides as potential substitutes for antibiotics ：GDP20 (RWRRWYRKFCR), CEN1HC-Br (FKKTFHKVSHAVKSGIHAGQRGCSA LGF), snake cathelicidin-BF (KFFRKLKKSVKKRAKEFFKKPRVIGVSIPF), tridecapepetide (CFRVCYRGICYRC), human cathelicidin LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE). According to our test, these five AMPs have shown excellent bactericidal efficiency against E. coli and P.acnes, which proves they possess great application prospects.
Figure 1 Five AMPs we collected
These AMPs were verified through two methods: plate inhibition zones assay and OD600 analysis. In plate inhibition zones assay, we placed pieces of paper soaked with AMP solutions onto plates inoculated with E.coli MG1655 and P.acnes and observing bacteria growth inhibition zones. In OD600 analysis, we added AMP solutions into liquid culture inoculated with the bacteria and measuring OD600 value.
Figure 2. AMPs test methods
To begin with, we tested the antimicrobial potencies of CEN1HC-Br and Snake cathelicidin-BF on E.coli MG1655 because they were the first AMPs synthesized.
CEN1HC-Br and Snake cathelicidin-BF's effects of killing E. coli MG1655 are weak as barely any difference can be seen between the plates with AMPs and those with water (Figures 3&4). However, in higher concentrations, significant inhibition zones of bacteria growth indicate the strong antimicrobial potencies of the two AMPs. When pieces of paper soaked with the AMP solutions are placed onto the plates, inhibition zones did not exceed the area covered by the paper. Therefore, the AMPs' range of bacteriacidal effect is limited within areas of direct contact with bacteria, which is good because it means that our product would not eliminate harmless microbes outside of acne infected regions.
Figure 3. CEN1HC-Br efficiency to E.coli MG1655 (plate).
Figure 4. Snake cathelicidin-BF efficiency to E.coli MG1655 (plate).
Based on previous results, we tested the antimicrobial potencies of GDP20, tridecapepetide, and human cathelicidin LL-37 on E. coli MG1655 only in high concentrations. All GDP20, tridecapepetide and human cathelicidin LL-37 demonstrated significant E. coli killing effects within areas of direct contact with the bacteria (Figure 5). Plates with tridecapepetide and human cathelicidin LL-37 had E. coli growing on the edges of the inhibition zones, indicating the effects of these AMPs are weaker.
Figure 5 GDP20, tridecapepetide, Human cathelicidin LL-37 efficiency to E.coli MG1655 (plate).
We then verified the 5 AMPs' effects of killing P. acnes with the same methods.
All 5 AMPs proved effective within direct contact with P. acnes. Determining from the size of inhibition zones, CEN1HC-Br is least effective in killing P. Acnes. The inhibition zone of tridecapepetide has a white background because the amino acids of the peptide's composition are strongly hydrophobic.
Figure 6. AMPs efficiency to P.acnes (plate).
We first verified the antimicrobial concentrations of CEN1HC-Br and Snake cathelicidin-BF as they are synthesized earliest. The AMP solution concentration gradient was initially set as 0, 4, 8, 12, 16mg/L, and tested their abilities to kill E. coli MG1655. After discovering significant difference between the results of the 2 AMPs, we adjusted the gradients of their concentrations. In later verification, we collected samples once in every 2 hours and measured OD600 for identification of antimicrobial potency.
Figure 7. CEN1HC-Br efficiency to E.coli MG1655 (OD600).
Figure 8. Snake cathelicidin-BF efficiency to E.coli MG1655 (OD600).
Though both are effective in inhibiting E. coli growth, CEN1HC-Br has weaker efficiency than Snake cathelicidin-BF. (Figures 7&8). E. coli reproduction nearly stopped when Snake cathelicidin-BF solution reached a concentration of 4 mg/L. However, it was almost the same as that in control group in CEN1HC-Br solution with concentration 16 mg/L. Therefore, in later verification we rearranged the concentration gradients of CEN1HC-Br and Snake cathelicidin-BF to 20, 24, 28, 32 mg/L and 0.5, 1, 2, 3 mg/L. Snake cathelicidin-BF proved to be greatly effective at 1mg/L while CEN1HC-Br's effect remains insignificant even at 32mg/L. Additionally, the efficient concentration of AMP solutions are lower in liquid culture because bacteria suspended in the solutions have better contact with AMPs.
We set the concentration gradient as 2, 4, 8, 12，16 mg/L for verification of GDP20, tridecapepetide, and human cathelicidin LL-37's antimicrobial potency on E. coli MG1655.
Figure 9. GDP20, tridecapepetide, human cathelicidin LL-37 efficiency to E.coli MG1655 (OD600)
The effects of all 3 AMPs peaked at 8 hours after cultivation. GDP20 and tridecapepetide proved to be most efficient at 16mg/L while human cathelicidin LL-37 displayed strongest inhibition at 4mg/L (Figure 9). It can be inferred that tridecapepetide has the strongest effect among the 3 AMPs.
We also tested the AMPs' abilities to kill P. acnes in liquid culture. Since P. acnes has a slow growth rate, we only measured OD600 after 48 hours of cultivation. In the results, tridecapeptide proved to be most efficient in killing P. acnes, followed by GDP20 and CEN1Hc-Br.
Figure 10. AMPs efficiency to P.acnes (OD600)
In conclusion, all 5 AMPs we collected are effective in eliminating E. coli and P. acnes.
