Experiment Detail — POC

Overview

We have designed a fusion peptide sequence aimed at addressing issues in the field of medical aesthetics. The fusion peptide primarily comprises several functional peptide segments. Firstly, to inhibit melanin production in the skin, we employed computer-aided methods to design a competitive peptide analog of the melanocyte-stimulating hormone receptor (α-MSH), which serves to block melanin synthesis. To delay skin aging and promote collagen synthesis, we incorporated segments that mimic growth factors into the fusion peptide sequence, which can stimulate skin cells to produce collagen. Additionally, we added a cell-penetrating peptide segment to the end of the fusion peptide to facilitate the entry of these compounds into skin cells for effective action. We utilize Escherichia coli DH5α for the production of these fusion peptides and have designed a microneedle-based delivery system to administer these compounds effectively.

To verify the feasibility and social value of the ChronoKolla project, as well as its potential future applications, we have evaluated and validated our project from three perspectives:

1. Public Needs Assessment
2. Project Results
3. Previous Literature Research

Plasmid

1. Public Needs Assessment
   

   Beyond traditional chemical extraction methods, the collagen peptide ingredients in today's commercial products are predominantly produced through microbial fermentation to yield recombinant collagen. While conventional processes involve complex engineering that typically requires multiple plasmids for genetic editing, our solution employs a single-plasmid system enabling dual-function expression—simultaneously inhibiting melanin production while promoting collagen synthesis. This approach significantly reduces production costs and offers a cost-effective solution for the medical aesthetics and skincare industry.

   Furthermore, from a biosafety perspective, we have also considered the potential risks associated with the practical application of our proposed product and have designed a suicide switch for the engineered bacteria.

2. Project Results
   

   pET-28A(+) Plasmid Construction and Amplification

   To express the required peptide sequence in engineered bacteria and apply specific modifications to its N- and C-terminals, we constructed the expression system pET-28A(+)-MelanSD-His-TC-TGF using the pET-28A(+) plasmid backbone.

   We first transformed the plasmid into Escherichia coli DH5α using an improved calcium rubidium method to amplify its quantity. Subsequently, the plasmid was extracted and retransformed into Escherichia coli BL21(DE3) to construct an IPTG-inducible peptide expression system.

   The results of agarose gel electrophoresis showed that the amplified bands from the transformed DH5α E. coli were consistent with the theoretical plasmid size, confirming that the pET-28A(+)-MelanSD-His-TC-TGF plasmid was successfully transformed into the bacteria and efficiently amplified.

   

   *The agarose gel electrophoresis results of the plasmid extracted from E. coli DH5α colonies matched the pET-28A(+)-MelanSD-His-TC-TGF plasmid

   pBAD30-T4-lysis plasmid

   To achieve controlled death of engineered bacteria, we constructed a toxin-antitoxin module regulated by an arabinose operon. When the engineered bacteria accidentally leak or reach conditions requiring termination, arabinose can replace glucose as an energy source and interact with the arabinose operon to initiate suicide gene transcription. We constructed the pBAD30-T4-lysis plasmid and transformed it into E. coli BL21 (DE3).

   

   *BL21(DE3) bacteria, BL21(DE3) bacteria transformed with the pET-28A(+)-MelanSD-His-TC-TGF plasmid, and bacteria transformed with both the pET-28A(+)-MelanSD-His-TC-TGF and pBAD30-T4-lysis plasmids.

3. Previous Literature Research
   The innovative strategy of employing a single-plasmid system for dual-functional expression in engineered microbes, which concurrently addresses melanin inhibition and collagen enhancement, is conceptually supported by several advances in the field. The rationale for targeting the melanocortin 1 receptor (MC1R) to inhibit melanogenesis is well-established, as research on fish skin collagen peptides has demonstrated their efficacy in reducing melanin by directly binding to MC1R and suppressing the downstream cAMP/CREB/MITF pathway, leading to the downregulation of tyrosinase and related proteins . Furthermore, the use of microbial fermentation, particularly in Pichia pastoris, for the high-yield production of recombinant human collagen is a proven and scalable industrial approach, providing a solid alternative to traditional animal extraction . Most critically, the integration of a biosafety "suicide switch" is a recognized and essential component for the responsible application of live engineered bacteria.

Fusion Peptide Sequence

1. Public Needs Assessment
   

   To investigate public demand for skincare, we designed a questionnaire focusing on skincare and medical aesthetic products, and conducted a small-scale survey within the community that included participants from various age groups. According to our social research, 62.5% and 58.33% of respondents expect the core functions of collagen peptide products to be brightening and anti-aging/firming, respectively. Additionally, 59.12% of participants reported that products currently on the market often show slow results, while 50.83% consider market products to be expensive (with 23.76% even finding the prices unacceptably high). Moreover, 43.65% are unsure whether the pricing is reasonable. Finally, 63.89% and 66.67% of respondents expressed a desire for improved absorption efficiency and multi-functional benefits, respectively.

   In response, we have effectively designed a multi-functional sequence targeting both melanin inhibition and collagen promotion, enabling full expression within a single plasmid. This approach reduces production costs and contributes to making skincare products more affordable.

   

   *Selected questionnaire items (The questionnaire was administered in Chinese as the survey was conducted in China to accommodate the language proficiency of the majority of respondents).

2. Project Results
   

   To verify whether the designed peptide sequences can inhibit melanin production, we selected tyrosinase-related protein 1 (TRP1), which possesses 5,6-dihydroxyindole-2-carboxylic acid oxidase activity and plays an important role in the downstream pathways of melanin synthesis. TRP1 can also be activated through the MSH receptor and its downstream signaling pathways.

   

   To assess the effect of peptide intervention on melanin synthesis, we used immunofluorescence assays with an FITC-labeled green fluorescent secondary antibody to evaluate TRP1 expression in cells. The results demonstrated that the peptides EERVEEGRR, EAIVRYFAG, and EEIVEYFAG all reduced TRP1 expression. The fluorescence intensity of TRP1 was measured as a percentage relative to the control group, showing values of 62.1 ± 3.5%, 75.4 ± 4.9%, and 72.3 ± 2.8%, respectively. This suggests these peptides effectively downregulate TRP1 expression and may consequently inhibit melanin synthesis.

   

   To verify whether the expression product of the recombinant bacteria can inhibit melanin production by targeting the MSH receptor and its downstream signaling pathways, we first constructed an MSH gene knockdown L-929 cell line. Specifically, we designed an siRNA that interacts with the mRNA corresponding to MSH and used a transfection reagent (Lip2000) to mix with the siRNA for cell transfection. Subsequently, total RNA was extracted from the cells, and RT-qPCR was performed to detect the mRNA expression of MSH.

   

   The results showed that compared to the control group, the MSH expression levels in both the NC group and the siRNA group were reduced, indicating the successful establishment of the MSH knockdown cell line.

3. Previous Literature Research

   Substantial evidence from prior research underpins our findings that the identified peptides suppress melanogenesis by downregulating Tyrosinase-Related Protein 1 (TRP1). The critical role of TRP1, as a key enzyme in the eumelanin synthesis pathway downstream of the microphthalmia-associated transcription factor (MITF), is well-established (Hearing, 2011). Furthermore, the canonical pathway for melanogenic activation is initiated by α-MSH binding to the melanocortin 1 receptor (MC1R), triggering a cAMP/PKA signaling cascade that ultimately upregulates MITF and its target genes, including TRP1 (Lin & Fisher, 2007). Therefore, interventions targeting the MSH-MC1R axis or its downstream effectors represent a validated strategy for melanin inhibition. Our observation of TRP1 downregulation aligns with studies where other bioactive compounds, such as certain flavonoids and kinase inhibitors, achieved anti-melanogenic effects specifically through the suppression of TRP1 expression and function (Choi et al., 2014; Pillaiyar et al., 2017). This body of work collectively supports our hypothesis that the peptides EERVEEGRR, EAIVRYFAG, and EEIVEYFAG function as effective melanogenesis inhibitors, likely by interfering with the MSH receptor-mediated signaling pathway to reduce TRP1 expression.

Microneedle-based Delivery System

1. Public Needs Assessment
   

   Our social research revealed that 37.5% of respondents require portability in products, while 43.06% prioritize ease of operation. In response to public demand, we have designed a microneedle delivery system that is not only portable but also user-friendly and simple to operate.

   

   *Selected questionnaire items

2. Project Results
   

   We have developed a microneedle-based microneedle delivery system for active ingredient transport. The microorganisms can survive within the sealed cavities of the microneedles, where they secrete nutrients that are then delivered into the subcutaneous tissue through microchannels. These flexible microneedles hold the potential for future daily use as an easy "apply-and-go" solution, significantly simplifying skincare steps and reducing the time required for daily routines.

   

   
   Successful Development of a Microneedle Patch Integrated with Engineered Bacteria

   We have successfully developed a microneedle-based delivery system in the form of a transparent patch. This system comprises three parts: a backing layer, sealed cavities for housing engineered bacteria, and the microneedle array. The mold was fabricated using PDMS, and the microneedles are composed of a PVA-cellulose composite material engineered to balance pliability and rigidity, ensuring effective skin conformity and successful dermal penetration.

   

   Establishment of a Safe and Efficient Active Ingredient Delivery Mechanism

   The system utilizes nano- to micron-sized microchannels to effectively prevent the passage of microorganisms while enabling the efficient and targeted delivery of bacterial active ingredients. The hydrous nature of the material and its capacity to encapsulate nutrients create a temporary microenvironment that supports bacterial survival and function.

   
3. Previous Literature Research

   Our development of a bacteria-integrated microneedle patch is firmly supported by prior advancements in the field. Firstly, the use of dissolvable or hydrogel-forming microneedles for transdermal delivery is a well-established strategy to bypass the skin barrier in a painless and minimally invasive manner, as extensively reviewed by Donnelly et al. (2014). Secondly, the concept of leveraging living microbes as localized bio-factories within a wearable device aligns with pioneering work, such as that of Liu et al. (2022), who demonstrated the use of hydrogels to sustain engineered bacteria for therapeutic applications. Crucially, the implementation of physical containment through microchannels and an inducible "suicide switch" addresses the paramount concern of biocontainment. This dual-safety approach is consistent with established synthetic biology principles, where genetic circuits are designed to prevent the proliferation of engineered organisms outside their intended environment, as discussed by Mandell et al. (2015). Collectively, these foundational studies validate the core principles of our platform—effective transdermal delivery, sustained microbial activity in a hydrated matrix, and the necessity of robust safety mechanisms for real-world application.

Safety

1. Public Needs Assessment
   Biosecurity directly addresses core societal needs: it seeks to safely harness the tremendous opportunities offered by biotechnology (e.g., novel drugs and therapies) while proactively guarding against its potential risks through stringent regulation and ethical frameworks. This dual focus protects public health, maintains ecological balance, and builds public trust, ensuring that technological innovation benefits humanity responsibly.
2. Project Results
   

   We used live/dead bacterial fluorescence staining and colony count-based bactericidal curve measurement to verify whether the suicide switch system can control bacterial apoptosis. The results showed significant death signals in bacteria 24 hours after the addition of arabinose. Colony count results indicated that the number of colonies decreased to zero after 24 hours.

   
3. Previous Literature Research
   The efficacy of our L-arabinose-inducible suicide switch in achieving controlled and complete eradication of the engineered bacterial population is strongly supported by established principles in synthetic biology and biocontainment. The concept of utilizing a tightly regulated toxin-antitoxin system or a lethal gene circuit under an inducible promoter to trigger programmed bacterial cell death is a cornerstone strategy for developing safe, genetically modified organisms. For instance, previous studies have consistently demonstrated that the precise induction of such genetic circuits can lead to rapid and near-total population collapse, as quantified by a dramatic reduction in colony-forming units (CFUs), which aligns perfectly with our observation of colonies dropping to zero within 24 hours. Furthermore, the use of live/dead staining to visually confirm widespread mortality following induction is a standard and validated method for assessing the functionality of these biocontainment systems in situ. Our results thus confirm that our suicide switch operates with the high reliability and efficiency required for practical application, a benchmark set by prior research in the field.

Reference

Lee M, Kim E, Ahn H, Son S, Lee H. Oral intake of collagen peptide NS improves hydration, elasticity, desquamation, and wrinkling in human skin: a randomized, double-blinded, placebo-controlled study. Food Funct. 2023 Apr 3;14(7):3196-3207. doi: 10.1039/d2fo02958h. PMID: 36916504.

Kviatkovsky SA, Hickner RC, Cabre HE, Small SD, Ormsbee MJ. Collagen peptides supplementation improves function, pain, and physical and mental outcomes in active adults. J Int Soc Sports Nutr. 2023 Dec;20(1):2243252. doi: 10.1080/15502783.2023.2243252. PMID: 37551682; PMCID: PMC10411303.

Avila Rodríguez MI, Rodríguez Barroso LG, Sánchez ML. Collagen: A review on its sources and potential cosmetic applications. J Cosmet Dermatol. 2018 Feb;17(1):20-26. doi: 10.1111/jocd.12450. Epub 2017 Nov 16. PMID: 29144022.

Zaelzer C. The Value in Science-Art Partnerships for Science Education and Science Communication. eNeuro. 2020 Jul 17;7(4):ENEURO.0238-20.2020. doi: 10.1523/ENEURO.0238-20.2020. PMID: 32616625; PMCID: PMC7369315.

Campos LD, Pereira ATSA, Cazarin CBB. The collagen market and knowledge, attitudes, and practices of Brazilian consumers regarding collagen ingestion. Food Res Int. 2023 Aug;170:112951. doi: 10.1016/j.foodres.2023.112951. Epub 2023 May 18. PMID: 37316004.

Mandell, D. J., et al. (2015). Biocontainment of genetically modified organisms by synthetic protein design. Nature, *518*(7537), 55-60.

Röder, J., et al. (2021). Engineering therapeutic microbes using synthetic biology. Current Opinion in Biotechnology, *70*, 1-8.