The project's success is demonstrated by showcasing our learning, design, experimental validation, and reflection on the product. It reflects our team's product iteration and development journey throughout the project's progression. Our team went through these four processes in three cycles, gradually completing the design of the biological hair conditioner.

Design

Hair damage can be triggered by environmental factors such as chemical treatment, heat, sunlight and pollution. Glutathione, as a natural antioxidant, can effectively neutralize free radicals and reduce free radical damage to hair. What's more, it can also act as a reducing agent, regulating disulfide bonds, thereby enhancing the elasticity and elasticity of hair (Ask, Magnus, et al.).

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Figure 1. Design of the gene circuit for gshF overexpression.
To produce glutathione, we overexpress GshF in E. coli. Specifically, we used pLac promoter (Lactose promoter) to express gshF in pSB1A3 plasmid (Figure 1), and the recombinant plasmid was transformed into E. coli BL21. Figure 2 shows the PCR results of the gshF gene. 

Figure 2. Gel electrophoresis of the gshF. The utilized DNA Marker is DL2000, which was procured from Takara in Japan. The bands, ordered from highest to lowest, correspond to DNA fragment sizes of 2000 base pairs (bp), 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

Test

Figure 3  Experimental results related to GshF.
To measure the production of Glutathione (GSH), the engineered strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C for 2 hours. The GSH content was determined using a reduced form glutathione (GSH) detection kit, and the results are shown in Figure 3A. Initially, the strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C. Samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of GshF on bacterial growth. The results are shown in Figure 3B. Additionally, different amounts of glutathione were added, and samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of glutathione on bacterial growth. The results are shown in Figure 3C. Furthermore, we investigated the effect of adding amino acid precursors (20 mM L-glutamic acid, L-cysteine, and glycine) on GSH production for 2 hours, and the results are shown in Figure 3D.

To determine the enzyme activity of GshF, we cloned its coding gene sequence into pET28a vector and transformed it into E. coli BL21. The engineered bacteria were then cultured overnight in LB medium, and 0.5 mM IPTG was added for induction. The next day, 1 g of bacteria was collected by centrifugation and resuspended in PBS (pH 7.4). The cells were then sonicated (150 W, 1 s on, 3 s off, for a total of 20 minutes) to obtain cell lysate. The reaction mixture in 10 mL PBS contained 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamine, 20 mM L-cysteine, and 20 mM glycine. After incubating at 37°C for 1 hour, the amount of reduced glutathione (GSH) was determined using a GSH assay kit, as shown in Figure 2E. 

The effect of oxygen on GSH production was also tested (2 hours), as shown in Figure 2F, by culturing the engineered bacteria in a CO2 incubator with O2 concentration adjusted to 0%, 20%, and 30%. After incubating at 37°C for 2 hours, the amount of GSH was measured using a GSH assay kit. The results indicated that the engineered strain significantly enhanced GSH production, with the highest yield at an O2 concentration of 20%.

Learn

After validation, it was found that the previous generation of the product already offers a certain degree of protection for the hair, and the results meet our preliminary expectations.

Design

 The primary focus of our study revolves around keratin, the protein that serves as the foundational component of hair. The α-keratin version of this protein has numerous chemical bonds, specifically known as disulfide bonds. These bonds play an essential role in shaping the elasticity and strength of hair. PepG, a peptide rich in cysteine residues, has the capacity to bond with keratin, hence contributing to the formation and repair of these vital disulfide bonds. 

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Instead of utilizing the previously explored chemical synthesis methods to derive PepG (as demonstrated by Cruz, Célia F., et al), our approach was bio-based, relying on the use of microorganisms. Microorganisms, due to their rapid growth and substantial yield capabilities, serve as an efficient means of producing proteins and peptides. A significant advantage of employing these microorganisms is their ability to secrete the peptides they produce, making collection straightforward. Due to the small size of PepG, it cannot be expressed in Escherichia coli independently (iGEM19_Manchester). Our chosen method involved the synthesis and cloning of a fusion expression gene into pET28a, with E. coli BL21 functioning as the host for the expression of the GshF and PepG fusion protein. To obtain PepG, thrombin recognition sites are added between the two protein sequences (Figure 4).

Figure 4. Fusion expression of PepG and GshF proteins. *tcs：Thrombin cleave site. (A) Gel electrophoresis of the  GshF-pepG. (B) PepG and GshF are linked through a Thrombin  site. The utilized DNA Marker is DL2000, which was procured from Takara in Japan. The bands, ordered from highest to lowest, correspond to DNA fragment sizes of 2000 base pairs (bp), 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

Test

 The fusion expression gene was synthesized and cloned into pET28a. Subsequently, E. coli BL21 was used as the host to express the fusion protein of GshF and PepG. The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The bacteria sediment was collected by centrifugation the next day and resuspended in 20 mM Tri-HCl buffer (containing 150 mM NaCl, pH 8.0), followed by sonication (150 W, 1s on, 3s off, for a total of 20 minutes) to obtain crude enzyme extract. The protein concentration of the crude enzyme extract was determined using the Bradford assay kit (Solarbio, China). At 25°C, 100 μg of crude enzyme extract was digested with 2U thrombin for 4 hours. The digestion products were mixed with 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamine, 20 mM L-cysteine, and 20 mM glycine, and incubated at 37°C for 1 hour. The content of reduced glutathione (GSH) was measured using a GSH detection kit. The results showed a significant increase in GSH content in the engineered bacteria (Figure 5A).

Figure 5. Fusion protein testing. (A) GshF enzyme activity test of cleaved products (B) Before and after comparison chart of using biological hair conditioner.
Subsequently, we proceeded with hair repair using hot-permed hair. Hair was washed with a 0.5% SDS solution, continuously stirred for one hour, and then allowed to air dry. Following this, a portion of the hair sample was secured with adhesive, curled with a clamp, and forcibly straightened to ensure that the hair remained free of any distortion. Afterward, the hair was incubated with the aforementioned cleavage products at 37°C for one hour. Following treatment, the hair was rinsed with distilled water and allowed to air dry naturally. As illustrated in Figure 5B, our finding demonstrated that the cleavage products were successful in restoring the quality of hair.  

Learn

This project not only opens up avenues for sustainable peptide production using microorganisms but also provides a promising approach to hair care and repair. It's worth noting that these cleavage products are produced by microorganisms, making them a sustainable solution that can be scaled up for hair repair. In comparison to traditional hair repair methods that often rely on harmful chemicals, our approach reduces the dependence on such substances, thereby lowering potential harm to both hair and scalp. This research contributes to improving overall hair health, making hair smoother, softer, and more manageable—particularly beneficial for those with damaged hair or in need of specialized care.

 

Design

The pigment responsible for hair color in organisms is melanin. We hope to darken hair by promoting melanin synthesis. The Tyr gene encodes a tyrosinase enzyme, which can convert tyrosine into dopa, and then further transform it into dopaquinone. Dopaquinone can subsequently be converted into melanin (Pavan, María Elisa, Nancy I. López, and M. Julia Pettinari).

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We overexpressed Tyr in E. coli BL21 using pSB1A3 plasmid, allowing it to synthesize tyrosinase by lac promoter (Figure 6 and Figure 7). This enzyme, in turn, promotes the darkening of the hair.

Figure 6. Design of the Try expression  system.

Figure 7. Gel electrophoresis of Try encoding gene. The utilized DNA Marker is DL2000, which was procured from Takara in Japan. The bands, ordered from highest to lowest, correspond to DNA fragment sizes of 2000 base pairs (bp), 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp, respectively.

Test

We initially conducted a validation study on the relationship between melanin content and the substance's light absorption capability (using light with a wavelength of 400nm for the experiment). The results are shown in Figure 8A. It is evident that the more melanin content there is, the stronger the substance's light-absorbing capability. This means that by increasing the melanin content in hair, we can enhance its light absorption ability, making it appear "blacker."

Next, we tested the change in melanin production by the engineered bacteria over time, ensuring its capability to produce melanin and gauging how long it would be appropriate to let the bacteria work. We found that the melanin production increased rapidly within the first 12 hours, after which the growth of melanin content slowed down. By 24 hours, the melanin production was only slightly greater than it was at the 12-hour mark and virtually stopped increasing after that.  

Figure 8. Experimental results related to melanin.
Subsequently, we attempted to research under which environmental conditions the melanin synthesis is maximized.First, we studied the impact of temperature on the GSH synthesis quantity in Figure 8C.  We observed that as the temperature decreased from 47°C to 25°C, the melanin yield over the same duration displayed an initial increase followed by a decrease. The maximum production was achieved at 37°C.

We also studied the effect of pH on melanin production in Figure 8D. We found that as the conditions shifted from neutral to acidic, the melanin yield gradually decreased. This suggests that the most optimal pH for melanin synthesis is around neutral.

Learn

The temporal analysis of melanin production underscores the significance of the initial 12 hours, during which the most substantial melanin production takes place. This insight has enabled us to fine-tune the timeframe of the bacterial synthesis process, ensuring both efficiency and cost-effectiveness. Regarding environmental conditions, our experiments have established that the optimal temperature for melanin synthesis stands at 37°C. In terms of pH, a neutral pH environment proves to be the most conducive for maximizing melanin production. In summary, the insights gained from the testing phase have provided invaluable guidance for refining our approach, ultimately leading us closer to our goal of enhancing melanin synthesis to promote hair darkening.

Through three sets of experiments, we successfully constructed three systems to provide hair-repairing functions for our shampoo. We produced short peptides containing cysteine from common substances in organisms to repair disulfide bonds. We also produced substances containing glutathione to enhance the repairing capability of the former. Additionally, melanin was synthesized step by step through tyrosinase.

1. Ask, Magnus, et al. "Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials." Microbial cell factories 12.1 (2013): 1-10.
2. Pavan, María Elisa, Nancy I. López, and M. Julia Pettinari. "Melanin biosynthesis in bacteria, regulation and production perspectives." Applied Microbiology and Biotechnology 104 (2020): 1357-1370.
3. Cruz, Célia F., et al. "Changing the shape of hair with keratin peptides." RSC advances 7.81 (2017): 51581-51592.