Unveiling Market Potential and Driving Forces
Since 1975, China's production of shampoo and conditioner has seen a remarkable surge, as evidenced in our data tables. However, this isn't just a statistical shift; it signifies a burgeoning public focus on hair-related issues, making hair quality an imperative concern.

Conditioner's Meteoric Rise: Beyond Numbers
Looking back, conditioner production skyrocketed from 0.8 thousand tons in 1975 to 25 thousand tons in 2000. This surge not only reflects a heightened demand for hair care products but, more importantly, underscores the colossal potential of the global hair care market. The catalyst behind this market expansion is the mounting attention individuals are giving to hair-related matters. As living standards improve, personal image and beauty care have gained prominence, with hair playing a pivotal role in shaping one's overall appearance.

Here is a table outlining common hair quality issues that people often experience:

These are some of the common hair quality issues that people may encounter, and addressing them often requires specialized hair care products and routines.

Yet, most conditioners on the market contain chemical agents, such as sulfates, silicones, and petroleum products, posing significant environmental risks. Sulfates, for instance, can lead to water acidification, adversely affecting aquatic life. Non-biodegradable silicones can accumulate in the environment, potentially harming aquatic ecosystems. Similarly, petroleum-based products like mineral oil are persistent pollutants, accumulating in water sources and posing threats to aquatic ecosystems. This raises a critical question: Do we truly require an abundance of chemical agents to nurture our hair? Is there a healthier, eco-friendlier approach to enhance hair quality?

This inquiry led us to introduce the concept of a biological conditioner—a solution that harmonizes technology and nature, harnessing bioactive substances like cysteine, glutathione, and tyrosinase to elevate hair quality. Our biological conditioner reduces reliance on chemical agents, rendering it more environmentally responsible while delivering superior hair repair effects.

The rapid growth of the global shampoo and conditioner market mirrors society's concern for hair issues and the prominence of hair quality problems in modern life. As technology and research advance, we anticipate continued market prosperity, offering increasingly innovative and effective hair care solutions. We firmly believe that through our biological conditioner, we can meet the challenges of this trend while safeguarding our environment and well-being, achieving genuine sustainable beauty.

Our project's overall design consists of three main genes: a repair system rich in cysteine peptides (pepG), an antioxidant system featuring Glutathione synthetase (GshF), and a coloring system with tyrosinase.

Because pepG is quite short, consisting of only 10 amino acids, it cannot be expressed independently in Escherichia coli ((iGEM19_Manchester)). Previous approaches involved chemical synthesis to obtain pepG. 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.

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.

This way, after expressing the fusion protein, we can use thrombin to cleave pepG and GshF, allowing them to function independently.

The pepG peptides are a specific type of peptide capable of binding to particular regions of hair proteins. These peptides engage with α-keratin and keratin proteins found in hair, resulting in alterations to the hair's shape, texture, and curliness, while also improving its hydrophilicity and softness. Notably, these peptides contain a significant amount of cysteine.

（This image derived from Part:BBa K2906100 - parts.igem.org ）
Cysteine is a sulfur-containing amino acid that plays a key role in hair restoration. First and foremost, it is a crucial component of our hair's primary protein—keratin—which gives hair its structure and strength.
In particular, cysteine contains sulfur elements that can form what are known as disulfide bonds. These bonds act as a bridge in the hair, connecting protein chains together to form a more stable structure. When our hair undergoes chemical treatments (like dyeing, perming), physical damage (like blow-drying, curling), or environmental damage (like UV radiation, pollution), these disulfide bonds can be destroyed, making the hair brittle and dry.

At this point, cysteine can "repair" these broken disulfide bonds, helping restore hair's strength and resilience. By supplementing sulfur elements, it helps reform disulfide bonds, thereby repairing damaged hair.
Furthermore, cysteine has antioxidant properties that can help protect hair from damage by free radicals, which are molecules that can cause hair aging and damage.

Glutathione (GSH)——a powerful antioxidant molecule naturally present in cells, known for its ability to combat oxidative stress and protect against damage caused by free radicals. GSH plays a crucial role in maintaining overall cellular health.
In the realm of hair care, GSH offers several advantages:

Glutathione's antioxidant properties make it a valuable component in various hair care products aimed at promoting healthy and vibrant hair.

2.2.2.Glutathione (GSH) is synthesized in cells through a two-step enzymatic reaction facilitated by the enzyme Glutathione synthetase, which is often abbreviated as GshF. 

Step 1: Formation of γ-Glutamylcysteine
1. In the first step, the enzyme GshF catalyzes the condensation of two precursor molecules: glutamate and cysteine.
2. This reaction forms a dipeptide known as γ-glutamylcysteine (γ-GC).
Step 2: Addition of Glycine
1. In the second step, GshF catalyzes the addition of a molecule of glycine to γ-GC.
2. This reaction results in the formation of Glutathione (GSH), which is the final product of the biosynthesis pathway.

Figure. The simplified pathway of reduced glutathione (GSH) synthesis. γ-GCS = γ-glutamylcysteine synthetase; GS = GSH synthetase; GshF = bifunctional GSH synthetase; GPx = GSH peroxidase; GR = GSH reductase; GSSG = GSH disulfide. Minus sign (−) denotes feedback inhibition. 

Tyrosinase plays a crucial role in the biosynthesis of Melanin. This process mainly includes the following steps:
· Oxidation of Tyrosine: In this step, Tyrosinase catalyzes the oxidation of Tyrosine to DOPAquinone. In this process, the phenolic hydroxyl group on one benzene ring of Tyrosine is oxidized to generate DOPAquinone.
· Conversion of DOPAquinone: DOPAquinone spontaneously transforms into precursors of melanin: DOPA and Dopachrome.
· Rearrangement of Dopachrome: Dopachrome undergoes a rearrangement reaction under the catalysis of Tyrosinase, resulting in the production of Dihydroxyindole carboxylic acid (DHICA) and Dihydroxyindole (DHI).
· Formation of Melanin: Finally, DHICA and DHI are oxidized and polymerized under the action of enzymes, forming Melanin.

Our innovative project incorporates three distinct, yet interconnected systems that address various aspects of hair care. From restoration to antioxidation, to color enhancement, these systems leverage the properties of key biological compounds—cysteine, glutathione, and tyrosinase, respectively—to deliver optimal results. Each system acts as a comprehensive solution to a specific hair concern, all while working harmoniously to promote overall hair health and aesthetics.

Target Users:
Our target users are consumers with damaged hair.

Production Process:
Our engineered bacteria will be cultivated through fermentation in a factory to produce Glutathione and Melanin. Subsequently, ultrasonic disruption will be used to break down the bacteria. The crude enzyme solution will be collected, and thrombin will be added to cut and release pepG. All these components will be collected and formulated into a liquid hair conditioner.

Usage Instructions:
After purchasing the hair conditioner, consumers only need to apply it thoroughly after shampooing. PepG will repair damaged cysteine, glutathione will enhance hair's antioxidant properties, and melanin will help maintain hair's dark and vibrant appearance.

Safety Precautions:
For reasons of biosafety, we have abandoned the idea of directly applying the engineered bacterial strain to human hair. Instead, we will produce the hair conditioner product in a factory for direct consumer purchase. The biological hair conditioner will undergo thorough bacterial disruption and stringent filtration before leaving the factory to ensure that the products sold to consumers do not contain any engineered bacterial strains.

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