In our school, there are many students who suffer from acne. Often you can see some students walking alone behind the crowd wearing masks. "I don't quite dare to talk to them because I'm afraid they will laugh at the acne on my face." said a student who was willing to be interviewed. Students between the ages of 14 to 18 are in a time when they are striving for beauty in their appearance, and this quest for beauty in turn triggers appearance anxiety. However, surveys have shown that this age group has the highest rate of acne of any age group, with 12 out of every 100 people having acne. Further some severe acne could affect patients for their entire lifetime, reducing the patient’s satisfaction with their appearance, inducing social barriers, causing psychological distress, and adversely affecting the individual’s study, work, and daily life. After our team's discussion, we decided to develop a treatmentfor acne to alleviate young people's appearance anxieties.

Acne Vulgaris, commonly referred to as acne, is a chronic inflammatory skin disorder that occurs due to the blockage of hair follicles with sebum - an oily substance that prevents the skin from drying out - and dead skin cells, resulting in the development of whiteheads, blackheads, and pimples [1].

Acne is associated mostly with Propionibacterium acnes (P. acnes) which releases extracellular enzymatic products that degrade facial sebum into long-chain fatty acids that cause inflammation [2].

Acne Vulgaris is one of the most prevalent dermatological conditions, particularly among teenagers aged between 15 and 19 years. It is estimated that almost 94-95% of pubertal individuals experience acne vulgaris at some point in their lives. [3]. Despite being a common condition, the incidence of acne vulgaris has been consistently increasing over the past few decades, necessitating greater attention to the issue [4].

However, despite the high prevalence of Acne Vulgaris, current medications are not effective enough and have various side effects that might further damage the skin.

The following are some of the most commonly used medications for acne and their corresponding downsides:

Vitamin A drugs: Our current understanding of Vitamin A drugs is inadequate, and the extent of its drug resistance is still unclear.

Retinoid: A type of vitamin A derivative. When applied in large concentrations, it could have a corrosive effect on the skin and lead to severe facial injuries.

Antibiotics: Studies have shown that P. acnes bacteria have a high resistance to antibiotics and antibiotics are not recommended as a single treatment for acne.

Peroxides: Applying too much of these medications may cause swelling and peeling of the skin.

Fig. 3. A commercially available drug that contains vitamin A

Our team has identified three main issues with current acne treatments:

1. Immature treatments with unclear drug side effects.

2. Significant side effects that can cause secondary skin damage.

3. Treatments that do not account for skin repair and nursing.

To address these issues, we have developed Acnedote, which provides effective acne treatment while avoiding the above-mentioned problems.

Problem 1: Bacterial colonization

Excess sebum is a key factor in acne formation. In acne patients, sebum can lead to hyperkeratinization of hair follicles. The exfoliation of sebum and keratin substances from the skin further clogs pores and triggers bacterial colonization.

Problem 2: Fatty acid accumulation

The antigens of the bacteria and the inflammatory substances they secrete will stimulate the exudation of inflammatory cells causing inflammatory lesions. In addition, lipases produced by P. acne hydrolyze triglycerides into pro-inflammatory free fatty acids, further exacerbating inflammatory lesions.

Fig. 5. Propionibacterium acnes mediates acne pathogenesis through innate immune activation

Dealing with facial acne can be a challenging experience that affects individuals from all walks of life. The physical symptoms of acne, such as redness and inflammation, can be distressing, but the psychological effects can be even more daunting.

Numerous studies have indicated that individuals experiencing facial acne often suffer from depression, anxiety, and low self-esteem [6, 7]. Those who suffer from it often feel embarrassed and self-conscious, leading to negative emotions and social isolation. It is important to raise awareness about the impact of acne on mental health and promote acceptance and empathy towards those who live with it.

The anti-inflammatory phase——Patch 1

Past studies revealed that P. acnes is highly associated with the formation acne for it releases extracellular enzymatic products that degrade facial sebum into LCFA that causes inflammation. Therefore, we sought to address the inflammation by simultaneously eliminating P. acnes and the LCFAs that they produce.

To eliminate P. acnes, we added a layer of silver ions to the patch. Silver ions can interfere with the normal functions of vital components that make up the cell of P. acne, including the cell wall and a wide spectrum of enzymes [8].

To remove the inflammatory LCFA from the skin, we engineered E.coli Nissle 1917 (EcN), a non-pathogenic strain of Escherchia. coli, to absorb and degrade LCFA from the face.

The healing phase——Patch 2

Through our interaction with different stakeholders such as patients and pharmaceutical manufacturers, we realized that simply addressing the inflammation would not be enough. What equally daunted patients as the inflammatory lesions were the marks that acne could leave behind. Therefore, we developed a second patch for patients to use after the first one. It can promote would healing and prevent scar formation by applying growth factors to the skin. EcN was engineered to produce Epidermal Growth Factors in this second patch.

The host organism that we selected for our patches is E.coli Nissle 1917 (EcN), a common microbe chassis. EcN is unique among E. coli strains due to its probiotic properties and non-pathogenic nature. It possesses several fitness factors that inhibit the growth of opportunistic pathogens, ensuring the safety of our product. Furthermore, EcN's gene expression is subject to extensive transcriptional and translational control, and its metabolic circuits are well-understood. These characteristics make EcN an attractive candidate for genetic engineering purposes.

Overview

For the first patch, we aimed to remove and degrade the inflammatory long-chain fatty acids (LCFA) from the skin by using an engineered strain of EcN. Moreover, we also sought to eliminate P. acnes from then thus removing the original cause of inflammation.

With these two aims in mind, we came up with the following designs.

Aim #1 – Remove and degrade the free fatty acids

P. acne releases lipases that degrade facial sebum into LCFA, which causes inflammatory lesions as a result. Therefore, we decided to design a bacterial metabolic pathway to remove and break down LCFA using the fadL and fadD genes. The cooperative function of the two genes is believed to complete the function of metabolizing LCFA [9].

Therefore, we adopted and improved the fadL + fadD system used for another purpose in our last year’s project.(https://2022.igem.wiki/rdfz-china/description)

The gene fadL codes for FadL, which is the long-chain fatty acid transport protein in E. coli. FadL is located in the outer membrane of E. coli. When bound with LCFA, FadL undergoes a conformational change which exposes the transport channel that facilitates LCFA transport into the periplasmic space (the space between the cell wall and the cell membrane) [9].

As for fadD, it codes for FadD, the only fatty acid Acetyl-CoA synthase (FACS) with a broad chain length specificity. FadD is responsible for the activation of beta-oxidation for the exogenous LCFA and the regulation of the transcription factor FadR, which optimizes fatty acid (FA) metabolism in response to environmental FAs [10, 11].

To wrap it up, LCFA first binds to FadL and undergoes a conformational change, allowing LCFA to cross the outer membrane of EcN. Once in the periplasmic space, LCFA becomes protonated and diffuses into the inner membrane. FadD then activates LCFA to CoA thioesters. After activation, LgoesA go through beta-oxidation and provides respiratory substrate.

In this year’s project, we made efforts to improve the accuracy of our results through adopting better extraction method quantitative measurements for LCFA. We also made better statistical interpretations of our results through the one-way analysis of variance.

Aim #2 – Eliminate pathogen

To further address the inflammation, we sought to address the root cause of inflammation – P. acnes, the LCFA producers.

A 2016 study reported that more than 50% of Propionibacterium acnes strains have developed antibiotic resistance, and the percentage could only be higher in 2023 [12]. Therefore, we turned to an alternative antibacterial agent that could not be discounted by antibiotic resistance – silver ions. Studies have shown that topical silver preparations are effective in common acne. This is because silver nanoparticles can bind to bacterial membrane proteins, thereby affecting membrane permeability. This method of bacterial inhibition produces low bacterial resistance, is not irritating to the skin, and protects the skin barrier [13].

Since current treatments do not consider skin repair, we sought to promote wound healing and prevent scar formation in the later stage of acne vulgaris by using growth factors for the skin.

We designed another engineered strain of EcN that has the capability of producing Epidermal Growth Factor (EGF) for the second patch.

Epidermal Growth Factor (EGF)

Epidermal growth factor is a single polypeptide composed of 53 amino acid residues that plays an important role in cell proliferation. It plays a crucial role in stimulating epithelial and fibroblast differentiation, enhancing collagen synthesis and supporting the wound healing process. Studies have proved that topical application of EGF is effective in improving acne lesions.

phoA: the tag for secretion

phoA is a signal peptide that guides newly synthesized proteins towards the secretion pathway.

Although EcN can slowly secrete the produced EGF, the secretion rate is relatively low, which is not efficient enough for acne treatment. Therefore, we decided to add a phoA secretion tag to the hEGF gene used for EGF production, facilitating the secretion of EGF into the periplasmic space of EcN.

The Signaling Pathway.

After translation, phoA binds to EGF to form the protoprotein. With the recognition and assistance of signal recognition particle (SRP), the protoprotein eventually attaches to the SecYEG complex, which is a translocon. In the translocon, phoA is dissociated from EGF and cleaved by signal peptidase, followed by the SecYEG complex, which mediates the translocation of EGF across the cell membrane for transmembrane delivery of EGF [15].

After being secreted outside the bacteria, EGF binds to the epidermal growth factor receptor (EGFR). This induce EGFR autophosphorylation and as a consequence activate its signal transduction cascade. This proves that EGFR modulating skin inflammation by affecting chemokine expression in keratinocytes.

Fig. 13. Model for protein targeting to the E.coli cytoplasmic membrane

Our cooperation with team Squirrel-Beijing 1 inspired us to use Bacterial Cellulose (BC) as the main material for our patches.

Bacterial cellulose is a friendly would dressing due to its non-toxicity and its potential to repress inflammation, regulate pH, promote tissue regeneration. Moreover, it has additional physical properties that make it suitable for wound dressing, such as its tensile strength, water retention, and relatively strong adhesion. Furthermore, BC is biocompatible, allowing for our engineered EcN to survive within the patch. Last but not least, BC is biodegradable and thus more environmentally friendly compared to alternative materials [16].

It is time to change the way we think about acne. Rather than hiding it away, we believe that acne is a natural part of puberty and should not be a source of shame. Unlike other beauty products that aim to conceal acne, we have designed a bold, colorful patch in the shape of a flower blossom to celebrate the unseen beauty of skin imperfections. Our message is clear: skin problems should not be stigmatized. For teenagers struggling with acne, these flower patches are a symbol of youth and growth that deserve to be embraced and cared for.

Our target audience comprises acne patients. To enable our consumers to use our product safely and effectively, we have designed a kit for this project. The kit includes: 1. Bacterial cellulose; 2. Silver ion solution; 3. Flower patches; 4. Engineered bacteria freeze-dried powder; 5. Biosafety processing box.

All of the above components will be manufactured in the factory. Specifically, our two-phase engineered bacteria will be produced in fermentation tanks and then processed into freeze-dried powder using freeze-drying technology to ensure the long-term viability of the engineered bacteria. Thanks to the high stability of all the components, our product does not require special storage conditions and can maintain its effectiveness for up to two years when stored at room temperature away from light. It will be packaged into kits and sold to consumers.

Once consumers purchase our product, it only requires a simple procedure for use. Consumers need to apply the silver ion solution on one side of the bacterial cellulose and apply the engineered bacteria on the other side, then cover it with the patch for use. To ensure that this process is error-free, we will also create instructional videos for consumers.

Additionally, we have developed an AI mathematical model that allows consumers to identify and precisely locate each acne area based on features such as color and texture. Subsequently, the severity of acne within each marked area will be graded as mild, moderate, or severe. Based on the assessment of each area, consumers can adjust the concentration of Escherichia coli accordingly (see the mathematical modeling section).

To ensure biological safety, during use, our engineered bacteria will be securely contained within the bacterial cellulose. After use, our provided biosafety processing box can be used to kill the engineered bacteria through high-temperature steam, preventing any gene leakage issues (see the safety section)."

Our team is developing engineered probiotic patches for acne treatment, with promising potential to provide an effective and user-friendly solution for acne management.

[1] H. C. Williams, R. P. Dellavalle, and S. Garner, "Acne vulgaris," (in eng), Lancet, vol. 379, no. 9813, pp. 361-72, Jan 28 2012, doi: 10.1016/s0140-6736(11)60321-8.

[2] K. A. Habeshian and B. A. Cohen, "Current Issues in the Treatment of Acne Vulgaris," (in eng), Pediatrics, vol. 145, no. Suppl 2, pp. S225-s230, May 2020, doi: 10.1542/peds.2019-2056L.

[3] I. L. Kanwar, T. Haider, A. Kumari, S. Dubey, P. Jain, and V. Soni, "Models for acne: A comprehensive study," (in eng), Drug Discov Ther, vol. 12, no. 6, pp. 329-340, 2018, doi: 10.5582/ddt.2018.01079.

[4] H. Chen, T. C. Zhang, X. L. Yin, J. Y. Man, X. R. Yang, and M. Lu, "Magnitude and temporal trend of acne vulgaris burden in 204 countries and territories from 1990 to 2019: an analysis from the Global Burden of Disease Study 2019," (in eng), Br J Dermatol, vol. 186, no. 4, pp. 673-683, Apr 2022, doi: 10.1111/bjd.20882.

[5] S. Moradi Tuchayi, E. Makrantonaki, R. Ganceviciene, C. Dessinioti, S. R. Feldman, and C. C. Zouboulis, "Acne vulgaris," (in eng), Nat Rev Dis Primers, vol. 1, p. 15029, Sep 17 2015, doi: 10.1038/nrdp.2015.29.

[6] R. G. Fried and A. Wechsler, "Psychological problems in the acne patient," (in eng), Dermatol Ther, vol. 19, no. 4, pp. 237-40, Jul-Aug 2006, doi: 10.1111/j.1529-8019.2006.00079.x.

[7] D. V. Samuels, R. Rosenthal, R. Lin, S. Chaudhari, and M. N. Natsuaki, "Acne vulgaris and risk of depression and anxiety: A meta-analytic review," (in eng), J Am Acad Dermatol, vol. 83, no. 2, pp. 532-541, Aug 2020, doi: 10.1016/j.jaad.2020.02.040.

[8] S. Chernousova and M. Epple, "Silver as antibacterial agent: ion, nanoparticle, and metal," (in eng), Angew Chem Int Ed Engl, vol. 52, no. 6, pp. 1636-53, Feb 4 2013, doi: 10.1002/anie.201205923.

[9] J. H. Bae, B. G. Park, E. Jung, P. G. Lee, and B. G. Kim, "fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity," (in eng), Appl Microbiol Biotechnol, vol. 98, no. 21, pp. 8917-25, Nov 2014, doi: 10.1007/s00253-014-5974-2.

[10] J. E. Cronan, "The Escherichia coli FadR transcription factor: Too much of a good thing?," (in eng), Mol Microbiol, vol. 115, no. 6, pp. 1080-1085, Jun 2021, doi: 10.1111/mmi.14663.

[11] T. J. Ford and J. C. Way, "Enhancement of E. coli acyl-CoA synthetase FadD activity on medium chain fatty acids," (in eng), PeerJ, vol. 3, p. e1040, 2015, doi: 10.7717/peerj.1040.

[12] T. R. Walsh, J. Efthimiou, and B. Dréno, "Systematic review of antibiotic resistance in acne: an increasing topical and oral threat," (in eng), Lancet Infect Dis, vol. 16, no. 3, pp. e23-33, Mar 2016, doi: 10.1016/s1473-3099(15)00527-7.

[13] J. Talapko, T. Matijević, M. Juzbašić, A. Antolović-Požgain, and I. Škrlec, "Antibacterial Activity of Silver and Its Application in Dentistry, Cardiology and Dermatology," (in eng), Microorganisms, vol. 8, no. 9, Sep 11 2020, doi: 10.3390/microorganisms8091400.

[14] H. K. Kim, I. K. Yeo, K. Li, B. J. Kim, M. N. Kim, and C. K. Hong, "Topical epidermal growth factor for the improvement of acne lesions: a randomized, double-blinded, placebo-controlled, split-face trial," (in eng), Int J Dermatol, vol. 53, no. 8, pp. 1031-6, Aug 2014, doi: 10.1111/ijd.12488.

[15] S. Pastore, F. Mascia, F. Mariotti, C. Dattilo, V. Mariani, and G. Girolomoni, "ERK1/2 regulates epidermal chemokine expression and skin inflammation," (in eng), J Immunol, vol. 174, no. 8, pp. 5047-56, Apr 15 2005, doi: 10.4049/jimmunol.174.8.5047.

[16] R. Portela, C. R. Leal, P. L. Almeida, and R. G. Sobral, "Bacterial cellulose: a versatile biopolymer for wound dressing applications," (in eng), Microb Biotechnol, vol. 12, no. 4, pp. 586-610, Jul 2019, doi: 10.1111/1751-7915.13392.

[17] Chen, H et al. “Magnitude and temporal trend of acne vulgaris burden in 204 countries and territories from 1990 to 2019: an analysis from the Global Burden of Disease Study 2019.” The British journal of dermatology vol. 186,4 (2022): 673-683. doi:10.1111/bjd.20882

[18]Moradi Tuchayi, Sara et al. “Acne vulgaris.” Nature reviews. Disease primers vol. 1 15029. 17 Sep. 2015, doi:10.1038/nrdp.2015.29

[19] Das, Shinjita, and Rachel V Reynolds. “Recent advances in acne pathogenesis: implications for therapy.” American journal of clinical dermatology vol. 15,6 (2014): 479-88. doi:10.1007/s40257-014-0099-z

[20] Ma, J., Lyu, Y., Liu, X. et al. Engineered probiotics. Microb Cell Fact 21, 72 (2022)

[21] Kim, Hyun Kyu et al. “Topical epidermal growth factor for the improvement of acne lesions: a randomized, double-blinded, placebo-controlled, split-face trial.” International journal of dermatology vol. 53,8 (2014): 1031-6. doi:10.1111/ijd.12488

[22] Valent, Q A et al. “The Escherichia coli SRP and SecB targeting pathways converge at the translocon.” The EMBO journal vol. 17,9 (1998): 2504-12. doi:10.1093/emboj/17.9.2504

[23] Pastore, S., Mascia, F., Mariotti, F., Dattilo, C., Mariani, V., & Girolomoni, G. (2005). ERK1/2 Regulates Epidermal Chemokine Expression and Skin Inflammation. The Journal of Immunology, 174(8), 5047–5056. doi:10.4049/jimmunol.174.8.5047

[24] Talapko, Jasminka et al. “Antibacterial Activity of Silver and Its Application in Dentistry, Cardiology and Dermatology.” Microorganisms vol. 8,9 1400. 11 Sep. 2020, doi:10.3390/microorganisms8091400

[25] Portela, Raquel et al. “Bacterial cellulose: a versatile biopolymer for wound dressing applications.” Microbial biotechnology vol. 12,4 (2019): 586-610. doi:10.1111/1751-7915.13392

[26] Volova, Tatiana & Shumilova, Anna & Shidlovskiy, Ivan & Nikolaeva, Elena & Sukovatiy, Aleksey & Vasiliev, A. & Shishatskaya, Ekaterina. (2018). Antibacterial properties of films of cellulose composites with silver nanoparticles and antibiotics. Polymer Testing. 65. 54-68. 10.1016/j.polymertesting.2017.10.023.