Team:Beijing 4ELEVEN/Proof Of Concept
Our team aims to produce a mixture of antimicrobial peptides (AMPs) and adhesive and cohesive proteins, which can be applied to the skin to kill P. acnes, one of the pathogens of acne vulgaris, and cure acne. Further into details, our project consists of the following two parts.
Part I: Construct effective AMPs expression systems.
Part II: Construct Adhesive/cohesive proteins and tyrosinase production system.
We selected 5 AMPs (CEN1HC-Br, GDP20, snake cathelicidin-BF, human cathelicidin LL-37, and tridecapeptide) as candidates through literature research. Firstly, we need to verify their antimicrobial potency. Thus we sent them to be synthesised directly and verified their effects of killing P. acnes and E.coli by plate inhibition zones assay and OD600 analysis. In plate inhibition zones assay,wo 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 1 Methods for verification.
In plate verification (Figure 2 &3), inhibition zones in all the plates except for the control plate indicate that all AMPs are capable of killing P. acnes and E. coli MG1655. While the Inhibition zones did not exceed areas of direct contact with AMP solution, which is good because it means that our product would not eliminate harmless microbes outside of acne infected regions.
Figure 2 AMPs efficiency to E.coli MG1655 (plate).
Figure 3 AMPs efficiency to P. acnes (plate).
In the OD600 verification, we set distinct concentrations gradients and viewed the growth curves of E. coli MG1655 for 12 h. Since P. acnes has a slow growth rate, we only measured OD600 after 48 hours of cultivation. The intuitive results are displayed on this page (Figure 4&5) , and detailed information can be found on the Results Page.
It can be inferred from Figure 4&5 that all 5 AMPs are effective in repressing the growth of E.coli and P.acnes. Interestingly, the efficiency of AMPs was quite different, Snake cathelicidin-BF proved to be the most prominent for E. coli almost stop growing when the its concentration reached only 4 mg/L. For P. acnes，GDP20 and Human cathelicidin LL-37 exhibited the most outstanding performance
Figure 4 AMPs efficiency to E.coli MG1655 (OD600).
Figure 5 AMPs efficiency to P. acnes (OD600).
All these results proved that our AMPs are effective in eliminating E. coli and P. acnes, thus indicating the feasibility of our project.
We inserted our AMP sequences onto pPIC9K plasmids, then transformed them into our chassis, Pichia pastoris, and cultivated the yeast cells in BMMY culture. AMPs expressed by Pichia pastoris is secreted and dissolved in the supernatant of the fermentation broth, which is then put through plate and OD600 verification.
Figure 6. Fermentation products efficiency to E.coli MG1655 (plate).
Figure 7. Fermentation products efficiency to P.acnes (plate).
The results of plate verification shows that the fermentation products' repression on both E. coli MG1655 and P. acnes growth is insignificant (Figures 6&7). Colonies grew into the drops of fermentation products. That may be caused by the low concentration of AMPs dissolved in the fermentation broth.
In OD600 verification, the fermentation exhibited significant antimicrobial potency. We tested fermentation broth's efficiency to both E. coli and P. acnes (Figures 8). It can be inferred from the results that the fermentation product is effective in repressing growth of both E. coli and P. acnes. Although the actual yield of AMPs is not measured, the verifications of antimicrobial potency supports the functionality of Pichia pastoris synthesized AMPs.
Figure 8. Fermentation products efficiency to E.coli MG1655 and P.acnes (OD600).
We integrated adhesive and cohesive proteins into our project design to help AMPs establish a long lasting effect on the skin. Based on the project of 2019iGEM Team Greatbay_SCIE, Stickit, we selected fp1-mfp5-fp1, CsgA-mfp5, and CsgA-mfp5-mfp5 recombinant proteins. However, the adhesive proteins' function require DOPA modification by tyrosinase, which can be achieved through two means: expressing adhesive/cohesive protein and tyrosinase separately, then conduct modification in vitro; or expressing both adhesive/cohesive protein and tyrosinase together trough constructing co-expression system in vivo. We explored these two processes.
In the in vitro system attempt, we successfully construct a RBS library and obtained high yield of tyrosinase (see details on our Improvement Page), while extracellular secretion of adhesive proteins in Pichia pastoris proved to be a failure (see details on our Results Page.)
Nonetheless, we successfully constructed the co-expression system of adhesive/cohesive proteins and tyrosinase.
In the in vivo system attempt, we introduced a cationic dye induced expression promoter, EilR-PJExD, discovered in the works of Thomas and his team, into our co-expression system (see details on our Engineering Page). The expression of adhesive/cohesive proteins were regulated by T7-LacI induced promoters. We were able to detect the successful expression of both proteins in our system (Figure 9).
Figure 9 Co-expression of adhesive/cohesive proteins and tyrosinase.
All the above results prove that we have successfully completed the two parts of our project, and also illustrate the scientificity and feasibility of our project.
