Hair is not only an important component of our appearance but also reflects our physical health. After delving into the structure and composition of hair, we discover that keratin, as the core protein of hair, plays a crucial supportive role. The numerous disulfide bonds within keratin are, in fact, the key chemical structures determining the form, texture, and strength of hair. With this knowledge in mind, we have specifically designed three biological systems aimed at providing comprehensive care and improvement for damaged and post-dye hair. 

Firstly, the Glutathione Antioxidant System addresses hair damage caused by chemical treatments, heat, and environmental factors such as sunlight and pollution. Glutathione, as a natural antioxidant, effectively neutralizes free radicals, reducing their harm to hair. More importantly, it also acts as a reducer, regulating disulfide bonds, thereby enhancing hair's elasticity and resilience. 

Secondly, the PepG Repair System features PepG, a unique decapeptide rich in cysteine. Due to its structure, it interacts with the disulfide bonds in keratin, aiding in the repair and strengthening of these bonds. In this way, PepG reinforces hair structure, making it healthier and shinier.

Lastly, the Tyrosinase Hair Coloring System has been developed to meet the demand for hair coloring. This system utilizes tyrosinase to produce melanin, providing a natural and healthy coloring method for hair, thus avoiding the damage caused by traditional chemical dyes to both hair and scalp. By combining these three systems, we aim to offer a safer and healthier biologically-based hair care solution, meeting the high standards of modern consumers for hair care.

To measure the production of Glutathione (GSH), the engineered E. coli BL21 that overexpress gshF based on pSB1A3 plasmid (Figure 1) 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 2A.

Figure 1. Design of the gene circuit for gshF overexpression and 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.
 
Then, 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 2B. 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 2C. 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 2D.

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%.

Figure 2. Experimental results related to GshF.

The primary component of hair is a protein called keratin, specifically in the form known as α-keratin. Keratin contains numerous chemical bonds called disulfide bonds, which are crucial in determining the shape, elasticity, and strength of hair. PepG is a short peptide rich in cysteine residues, which can bind to keratin, participating in the formation and repair of disulfide bonds, making hair straighter and softer (Figure 3). Previous approaches involved chemical synthesis to obtain PepG (Cruz, Célia F., et al). We intend to use microorganisms to produce this peptide. This is because microorganisms possess certain characteristics that make them well-suited for protein and peptide production: they can grow rapidly and yield high quantities. These microorganisms can automatically secrete the produced peptides, which is very convenient because it means we can easily collect the peptide products from the culture medium. However, it was previously confirmed that PepG could not be efficiently expressed in Escherichia coli due to its small size (iGEM19_Manchester).

Figure 3. The stucture of PepG, which full of cystine residues (Pink lable). This image derived from Part:BBa K2906100 - parts.igem.org)
 
We aim to express it in E. coli using synthetic biology methods.  To achieve this, we have innovatively fused pepG with Glutathione synthetase (GshF) for co-expression, with a Thrombin cleavage site linking the two gene segments（Figure 4）. Thrombin, known for its strong sequence-specific cleavage and high hydrolysis efficiency, is widely used in genetic engineering product development. One of its applications is as a protease tool for the specific cleavage of recombinant fusion proteins.  The optimal cleavage site for thrombin is X4-X3-P-R[K]-X1'-X2', where X4 and X3 are hydrophobic amino acids, and X1' and X2' are non-acidic amino acids.  The recognition site we are using is L-V-P-R-G-S.

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.
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 in Figure 5 .

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 (Figure 5B). The results indicated that the cleavage products effectively restored hair quality. 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.  

 
To produce melanin, we used pSB1A3 vector to overexpress Try enzyme in Escherichia coli (Figure 6). Try encoding gene is driven by plac promoter. In addition, we added B0015 as a terminator to the gene circuit. Subsequently, we transformed the recombinant plasmid into E. coli BL21 to construct the engineered strain (Figure 7).

Figure 6. 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. 

Figure 7. Design of the Try expression  system.
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."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. This observation can serve as a reference for subsequent, cost-effective synthesis decisions in Figure 8B.

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.

Figure 8. Experimental results related to melanin.

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. Cruz, Célia F., et al. "Changing the shape of hair with keratin peptides." RSC advances 7.81 (2017): 51581-51592.
3. 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.