Team:Beijing 4ELEVEN/Poster

Poster: Beijing_4ELEVEN


Acneutralizer
Poster by team Beijing_4ELEVEN 2020

Abstract and Introduction

Acneutralizer: Utilizing antimicrobial peptides (AMPs) and protein adhesives to treat acne vulgaris

Acne vulgaris troubles teenagers and sometimes adults as it not only affects the skin but also self-confidence. Traditional treatment to acne vulgaris involving antibiotics may trigger drug resistance in acne pathogenic bacteria, especially Propionibacterium. acnes. Antimicrobial peptides (AMPs) are considered excellent alternatives of antibiotics for their capability of providing effective, broad-spectrum defenses against infections and exhibiting relatively low potential to elicit resistance. However, AMP curing products nowadays are not abundant, and most AMPs are produced via chemosynthesis, a rather costly method incapable of yielding AMPs with great antimicrobial potency. Therefore, we aim to create an anti-acne product which consists of biosynthesized P. acnes killing AMPs, adhesive/cohesive proteins, and tyrosinase. The mixture which we named Acneutralizer forms a thin film when applied to acne-infected skin. This product may be brought to market, helping acne sufferers improve their conditions by easily killing P. acnes.

Authors and Our team

Yuting Yang1,3, Siyu Jiang1,4, Borong Liang1,4, Yihan Qi1,3, Yuchen Zhang1,3, Yuchu Wang1,3, Shizhuo Xu1,3, Jiawei Zhou1,4, Ruijuan Xiang2,5.

  • 1 iGEM Student Team Members
  • 2 iGEM Team Instructors
  • 3 Beijing No.4 High School International Campus
  • 4 Beijing National Day School
  • 5 Bluepha Lab, Beijing, China
    • Inspiration
    AMPs are great alternatives of antibiotics for their efficiency to kill various bacteria and unlikeliness to elicit resistance in microbes. AMPs such as GDP20, Human Cathelicidin LL-37, Tridecapeptide, kills P. acnes, the main pathogen of acne vulgaris. Pichia pastoris, a yeast species, can be used as chassis for biosynthesis for its ability to withstand the AMPs it produce due to its different cell structure to bacteria. Protein adhesives, consisting of mussel foot originated adhesive and cohesive proteins, form waterproof biocoating, which is great for keeping our AMPs in place on users' skin.

    Problem
    Acne vulgaris negatively affects its victims' appearance and confidence.Traditional acne treatment involving antibiotics triggers antibiotic resistance. AMPs are currently produced commercially via chemosynthesis, a costly and unefficient method. Common means of biosynthesis, though more cost-effective and capable of producing complex peptides, produce AMPs that kill the chassis bacteria and disturbs its expression. AMP solutions alone do not last long enough on the skin to function due to their strong fluidity.

    Objectives
    We aim:
    -To create an anti-acne AMP product that forms biocoating when applied to skin and cures acne by killing P. acnes.

    -To explore Pichia pastoris synthesis of AMPs and provide companies with inspiration for AMP biosynthesis.


    Methodology
    In order to test the antimicrobial potencies of our 5 selected AMPs (GDP20, Tridecapeptide, Snake Cathelicidin-BF, Human Cathelicidin LL-37, CEN1HC-Br), we used the following methods of verification.
    Plate Verification
    Drops of and pieces of paper soaked with AMP solutions are placed onto culture plates inoculated with P. acnes and E. coli MG1655. Formation of bacteria growth inhibition zones is indication of sufficient potency.














    OD600 Verification
    AMP solutions of different concentrations are added to liquid culture inoculated with P. acnes and E. coli. The OD600 values of bacteria in liquid culture is measured. Decrease of OD600 value indicates measureable antimicrobial potency of AMPs.
    Construction of a new inducible system
    2019 Greatbay_SCIE's intracellularly modified co-expression system involved only the inducible promoter T7-LacI, which made the expression of the two proteins impossible to be regulated separately. We introduced a new tightly repressible induction system. In the system, PJExD promoter is regulated by EilR repressor, and several cationic dyes act as efficient low-cost inducers. We used sfGFP for quantitative analysis of the system. During verification, seven different dyes induced the expression of sfGFP, and Crystal Violet (CV) proved to be the most efficient inducer.



    We tested the expression of tyrosinase using the new expression system with 1 μM CV as inducer. Correct band of tyrosinase appeared in SDS-PAGE gel results, and tyrosinase yield could reach 1.44 mg/mL.
    Part I. AMP Production and Verification

    Original AMP Antimicrobial Potency Verification

    Our 5 AMPs were synthesized directly and their efficiency to P. acnes and E. coli were tested through plate and OD600 verification.

    In plate verification, inhibition zones in all the plates except for the control plate indicate that all AMPs are effective to both P. acnes and E. coli MG1655. The AMPs' range of bacteriacidal effect is limited within areas of direct contact with bacteria, meaning that our product would not eliminate harmless microbes outside of acne infected regions.

    In OD600 verification with E. coli, we found that all 5 original AMPs are successfully inhibiting the growth of E. coli MG1655. While 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.

    In OD600 verification with P. acnes, All 5 original AMPs are effective in inhibiting growth of P. acnes. While GDP20 and Human cathelicidin LL-37 proved to be most efficient to P. acnes. The results in OD600 verification to E.coli and P.acens shows that the same antimicrobial peptide has different inhibitory effects on different strains.



    Pichia pastoris Synthesized AMP Antimicrobial Potency Verification

    We synthesized our AMPs by transforming plasmids containing their sequences into Pichia pastoris and inducing the expressions with methanol. Then we verified the fermentation products' antimicrobial potencies against P. acnes and E. coli using the two verification methods.

    In plate verification, the fermentation products' effects of killing E. coli MG1655 and P. acnes are not significant due to low volume of AMPs dissolved in the solutions. In OD600 verification, Snake Cathelicidin-BF and Human Cathelicidin LL-37 are most effective in killing E. coli and Tridecapeptide and Human Cathelicidin LL-37 are most effective in killing P. acnes.

    Although the actual yield of AMPs is not measured, the verifications of antimicrobial potency support the functionality of Pichia pastoris synthesized AMPs.




    Part II. Adhesive and Cohesive Proteins and Tyrosinase Production
    We improved 2019 Greatbay_SCIE's tyrosinase expression system by modifying its 6 base pairs of its RBS sequence TAAGTATAAGNNNNNNATAT. 15 RBSs are taken into our RBS library through RBS calculator verification of the modified RBSs.

    The expression of mTyr-CNK is verified with different RBSs at inducer concentration 0.5mM, 25℃, and induction time for 20 hours. The production of proteins controlled by different RBSs reached 2.94 mg/mL, which was more than 4 times higher than the control group of 0.70 mg/mL.


    Production of adhesive and cohesive proteins in Pichia pastoris

    We transformed plasmids containing fp1-mfp5-fp1 and CsgA-mfp5-mfp5 into Pichia pastoris GS115 for adhesives production. Adhesive and cohesive proteins in the fermentation supernatant are verified by SDS-PAGE.

    We did not observe any correct bands in gel results--no bands appeared in the results of fp1-mfp5-fp1, and a few bands way too large appeared in that of CsgA-mfp5-mfp5. This may be the result of the formation of inclusion bodies, or the expression of multiple genes together forming a complex folder.


    Construction of adhesive and cohesive proteins and tyrosinase co-expression system

    Since expressing the adhesive and cohesive proteins and tyrosinase separately did not prove to be successful, we constructed the adhesive/cohesive protein and tyrosinase co-expression system by transforming the adhesives expression plasmid and tyrosinase expression plasmid into E.coli BL21 together.

    The results are successful as correct bands appear in the SDS-PAGE gel.


    Part III. Future Directions
    We will:

    -Verify and improve the effects of AMPs and protein adhesives together as our final product.

    -Make the removing process of Acneutralizer unpainful with protease hydrolysis of adhesive coating.

    -Add more AMPs to expand Acneutralizer's range of effect.

    -Improve portability of Acneutralizer by applying PHA coating technology provided by NWU-CHINA-A.

    Human Practices
    Interviews of Teenagers with Acne

    We interviewed mainly teenagers about their experiences with acne and their anticipation of an effective anti-acne product, and learned that most of them are troubled by acne vulgaris' negative effects on their daily lives. They seek treatments that cure acne in a short time, and contain ingredients popularly known to have great effect on acne treatment, but are normally confused by the vast variety of treatments on the internet, not knowing which is most effective.



    Survey and Article on Public Awareness of Antibiotic Resistance

    We established an online survey gathering information about people's awareness of the negative effects of antibiotics misuse, and found out that they are very aware of the correct uses of antibiotics, and are willing to accept an innovation that reduces the worsening of antibiotic misuse in treatment of diseases including acne.

    We also called attention to the worsening of antibiotic resistance and introduced correct uses of antibiotics in our public account article.

    Interviews of Cosmetics Companies

    We learned about common acne treatments and the increasing demand for new anti-acne products and treatment. They encouraged the development of our product and suggested that the use of AMPs in acne treatment is very innovative.

    Interview of Dermatosis Doctor

    We interviewed the director of dermatosis at an UFH hospital. We learned about the types of acne pathogenic bacteria and their mechanism of infection; that the use of antibiotics in acne treatment has not triggered severe resistance so they need not be replaced by AMPs; that there had been a few attempts at using AMPs for acne treatment, but the results were not much better than the results of antibiotics; and that our product which targets only one among numerous pathogens of acne cannot be efficient.

    Development of Acne Diagnosis and Treatment Applet

    To further help people with acne, we developed an applet based on the 2019 Acne Treatment Guidelines of China, which provides acne diagnoses and gives treatment advice based on information input by its users. When we introduced the applet to acne patients via our wechat official account, we received measureable positive feedback for helping people who had not gone to hospitals gain a primary impression of their conditions and credible treatment advice.
    References and Acknowledgements
    References:
    [1] Hazarika, Neirita, and Radha K. Rajaprabha. "Assessment of life quality index among patients with acne vulgaris in a suburban population." Indian Journal of dermatology. 2016, 61, 163.
    [2] Boen, M; Jacob, C. A Review and Update of Treatment Options Using the Acne Scar Classification System. Dermatol Surg. 2019,45,411.
    [3] Layton AM. Disorders of sebaceous glands. In: Burns T, Breathnach S, Cox N, Griffiths C, editors. 8th ed. Oxford: Wiley-Blackwell Publication. 2010, 42, 1–89.
    [4] Jović, A.; Marinović, B.; Kostović, K.; Čeović, R.; Basta-Juzbašić, A.; Bukvić Mokos, The Impact of Psychological Stress on Acne. Acta dermatovenerologica Croatica : ADC,2017, 25(2), 1133–1141.
    [5] https://www.cdc.gov/drugresistance/index.html
    [6] O’Neill, J. Antimicrobial peptides: Tackling of a crisis for the health and wealth of nations.
    [7] Ramesh, S., de la Torre, B. G., Albericio, F., Kruger, H. G., & Govender, T. (2016). Microwave-Assisted Synthesis of Antimicrobial Peptides. Antimicrobial Peptides, 51–59. doi:10.1007/978-1-4939-6737-7_4
    [8] https://2019.igem.org/Team:Greatbay_SCIE
    [9] Bahar, A.A.; Ren, D. Antimicrobial Peptides. Pharmaceuticals 2013, 6, 1543-1575.
    [10] Hwang, Dong Soo; Zeng, Hongbo et al. Protein- and Metal-dependent Interactions of a Prominent Protein in Mussel Adhesive Plaques. The American Society for Biochemistry and Molecular Biology, Inc. 2010.

    Acknowledgements:
    Ruijuan Xiang: Instructor of our project.
    Nan Xiang: PI of our project.
    Qingyao Zhu: Advisor of our project.
    Heyuan Ni: Advisor of our project.
    Liwen Zhu: Advisor of our project.
    Mingxuan Li: Advisor of our project.
    Zhi Sun: Experimental suggestion and model guidance.
    Xuanyu Zhao: Experimental suggestion guidance.
    Gaoyan Wang: Experimental method recommendations.
    Simin Li: Yidu Cloud employee, applet tech guidance.