iGEM 2023 — Team bfsu-icunited

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{% block title %}Notebook{% endblock %}
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{% block detail\_title\_a1 %}Experimental preparation for week 1{% endblock %}
{% block detail\_content2 %}Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain
Experimental procedure:
1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack, Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.
2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2 boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it was cleaned up properly afterwards.
3. Activated strains: 1 tube of Escherichia coli DH5α/ empty vector 1, 1 tube of DH5α/ carrying try gene,1 tube of DH5α/ carrying gshF gene, and 1 tube of DH5α/ carrying GshF-pepG gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the orifice and plug of the test tube, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the orifice and plug of the test tube again. After sealing the tube, the tube was bundled and incubated on a shaker overnight (150 rpm, 37℃).
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{% block detail\_title\_a2 %}2 weeks to extract the templates, began to construct the plasmid{% endblock %}
{% block detail\_content3 %}Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR
Experimental procedure:
1. Extraction of plasmids: four kinds of plasmids were extracted according to the instructions of the plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.
2. Dilute the primers
The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water was added to dilute the primer to the appropriate concentration
3.PCR reaction:
(1) Target genes pheP, PAL and two kinds of vector DNA fragments were amplified by high fidelity enzyme Pfu. The reaction system is as follows:
Pfu 0.5 μL
5 μL of 10xPfu buffer
Primer 1 1 μL
Primer 2 1 μL
Template (plasmid extracted above) : 1μL
25μL dNTP Mixture
20 μL of sterilized water
(2) Perform the PCR amplification procedure
At 98 ° C for 5 min
The following 3-step reaction was performed for 30 cycles
98℃ 10s
55℃ 5s
72℃ 1min/kb
72℃ 8 min
The plasmids, primers and products required for PCR are shown in the table below:
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{% block detail\_title\_a3 %}3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling{% endblock %}
{% block detail\_content4 %}Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental procedure:
1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled.
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2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit
Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min, and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA concentration was determined using Nanodrop.
3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:
1 μg of DNA
1 μL of EcoRI
1 μL XhoI
4 μL 10xY buffer
H2O up to 20μL
The enzyme was digested at 37 ° C for 4h
4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.
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{% block detail\_title\_a4 %}At week 4, competent cells were prepared{% endblock %}
{% block detail\_content6 %}Experimental plan: E. coli DH5α and Rosetta were activated to prepare competent cells
Experimental procedure:
1. Activated strains: including E. coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper, connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube stopper again. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).
Experimental procedure:
2. Preparation of competent cells:
(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a refrigerator at 4 ° C.
(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard, and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again (4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.
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{% block detail\_title\_a5 %}At week 5, the cells were connected and transformed{% endblock %}
{% block detail\_content7 %}Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform ligation products
Experimental procedure:
1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).
2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment volume ratio can also be calculated according to the instructions) :
T4 DNA ligase 1 μL
2 μL of 10 x ligation buffer
1 μL of Linear plasmid
Insert 6 μL of DNA
Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was stored at -20 ° C.
3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1 mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100 μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.
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{% block detail\_title\_a6 %}The successful plasmid was extracted and expanded at the 6th week{% endblock %}
{% block detail\_content8 %}Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation
Experimental procedure:
1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as templates for colony PCR using Taq enzyme. The primers are TF, TR. The reaction system and procedures were carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed
2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp antibiotic was added to expand the culture.
3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.
4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.
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{% block detail\_title\_a7 %}The standard curve and content of melanin were established at the 7th week{% endblock %}
{% block detail\_content9 %}Experimental procedure:
Different concentrations of melanin were added to LB medium, and the absorbance at 400nm was measured and recorded. Establishing a standard curve
LB medium was supplemented with 50 mg/L L-DOPA and 8 mg/L L-tyrosine as substrates, and bacteria were grown at 37°C on a shaker (180rpm). Every 4 hours, 2mL of the bacterial solution was transferred to the EP tube, the cells were broken up and centrifuged, and the absorbance at 400nm was measured and recorded. The production curve was measured within 24h.
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{% block detail\_title\_a8 %}The effect of temperature, PH on melanin production was measured at week 8 {% endblock %}
{% block detail\_content11 %}Experimental procedure:
Bacteria were grown at 37°C and 25°C on a shaker (180rpm). OD400 was measured at 24h and the melanin concentration was converted from a standard curve. The pH of LB medium was adjusted to 7,6,5 using hydrochloric acid. Bacteria were grown in a shaker (180rpm) at 37 ° C. OD400 was measured at 24h and the melanin concentration was converted from a standard curve.
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{% block detail\_title\_a9 %}Glutathione production at week 9, effects on bacterial growth, and effects of precursors and oxygen on GSH production{% endblock %}
{% block detail\_content13 %}Experimental procedure:
To measure GSH production, strains were resuspended in LB medium to an OD600 of 0.1 and incubated for 2h at 37°C. The GSH content was determined by the reduced glutathione (GSH) content detection kit. Initially, the strains were resuspended in LB medium to an OD600 of 0.1, incubated at 37°C, and sampled at 4, 6, 8, and 12 for OD600 measurement to determine the effect of GshF on bacterial growth. Wild type E. coli was resuspended in LB medium to OD600 of 0.1, incubated at 37°C, and different amounts of glutathione were added. Samples were taken at 4, 6, 8, and 12h to determine the effect of OD600 and glutathione on bacterial growth. The recombinant vector was transformed into E. coli BL21 cells, and positive clones were screened on LB AGAR plates containing ampicillin. The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The next day, 1 g of bacteria was collected by centrifugation, resuspended in PBS (pH 7.4), and cells were then lysed by sonication (150 W, 1s sonication, 3s interval, for a total of 20 min) to obtain cell lysates. The 10 mL PBS reaction mixture contained 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamate, 20 mM L-cysteine, and 20 mM glycine. After 1 h of reaction at 37°C, the amount of GSH was determined using a reduced glutathione (GSH) content assay kit. The tested engineered bacteria were statically cultured in a CO2 incubator (O2 concentration was adjusted to 0%, 20% and 30%). After incubation at 37°C for 2h. The content of GSH was determined by reduced glutathione (GSH) content detection kit.
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{% block detail\_title\_a10 %}Fusion expression and thrombin cleavage of GshF-PepG at week 10{% endblock %}
{% block detail\_content15 %}Experimental procedure:
The engineered bacteria were cultured overnight in LB medium. 0.5 mM IPTG was added for induction. The bacterial precipitate was collected by centrifugation the next day, resuspended in 20 mM Tri-HCl buffer (containing 150 mM NaCl, pH 8.0), and then sonicated (150 W, 1s sonication, 3s interval, for a total of 20 min) to obtain the crude enzyme solution. The protein concentration of the crude enzyme solution was determined using the Bradford Assay kit (Solarbio, China). 100 μg of the crude enzyme solution was digested using 2U thrombin for 4h at 25 ° C. 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamic acid, 20 mM L-cysteine and 20 mM glycine were added to the cleavage product, and the reaction was carried out at 37 ° C for 1 hour. The GSH content was determined using the GSH content detection kit.
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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5004/wiki/cover/engineering.jpg{% endblock %}
{% block detail\_content1 %}{% endblock %}
{% block detail\_title\_a1 %}Overview{% endblock %}
{% block detail\_content2 %}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.
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{% block detail\_img1 %}https://static.igem.wiki/teams/5004/wiki/engi/img1.png{% endblock %}
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{% block detail\_title\_a2 %}Cycle 1: Enhancing Hair Repair-Antioxidant system{% endblock %}
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{% block detail\_title\_b1 %}Design{% endblock %}
{% block detail\_content5 %}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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{% block detail\_title\_b2 %}Build{% endblock %}
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{% block detail\_img2 %}https://static.igem.wiki/teams/5004/wiki/engi/img2.png{% endblock %}
{% block detail\_content7 %}

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.
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{% block detail\_img3 %}https://static.igem.wiki/teams/5004/wiki/engi/img3.png{% endblock %}
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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.

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{% block detail\_title\_b3 %}Test{% endblock %}
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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%.
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{% block detail\_title\_b4 %}Learn{% endblock %}
{% block detail\_content11 %}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.
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{% block detail\_title\_a3 %}Cycle 2: Enhancing Hair Repair-Disulfide bond repair{% endblock %}
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{% block detail\_title\_b5 %}Design{% endblock %}
{% block detail\_content13 %} 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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{% block detail\_title\_b6 %}Build{% endblock %}
{% block detail\_content14 %}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).
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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.

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{% block detail\_title\_b7 %}Test{% endblock %}
{% block detail\_content16 %} 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).
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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.
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{% block detail\_title\_b8 %}Learn{% endblock %}
{% block detail\_content18 %}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.
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{% block detail\_title\_a4 %}Cycle 3: Assisting in Hair Dyeing{% endblock %}
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{% block detail\_title\_b9 %}Design{% endblock %}
{% block detail\_content20 %}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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{% block detail\_title\_b10 %}Build{% endblock %}
{% block detail\_content21 %}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.
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Figure 6. Design of the Try expression system.

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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.

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{% block detail\_title\_b11 %}Test{% endblock %}
{% block detail\_content24 %}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.
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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.
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{% block detail\_title\_b12 %}Learn{% endblock %}
{% block detail\_content26 %}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.
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{% block detail\_title\_a5 %}Conclusion{% endblock %}
{% block detail\_content27 %}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.
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{% block detail\_title\_a6 %}References{% endblock %}
{% block detail\_content28 %}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.
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[{{self.detail\_title\_b3()}}](#b3)
[{{self.detail\_title\_b4()}}](#b4)
[{{self.detail\_title\_a3()}}](#a3)
[{{self.detail\_title\_b5()}}](#b5)
[{{self.detail\_title\_b6()}}](#b6)
[{{self.detail\_title\_b7()}}](#b7)
[{{self.detail\_title\_b8()}}](#b8)
[{{self.detail\_title\_a4()}}](#a4)
[{{self.detail\_title\_b9()}}](#b9)
[{{self.detail\_title\_b10()}}](#b10)
[{{self.detail\_title\_b11()}}](#b11)
[{{self.detail\_title\_b12()}}](#b12)
[{{self.detail\_title\_a5()}}](#a5)
[{{self.detail\_title\_a6()}}](#a6)

```
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```

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/5004/wiki/cover/common.jpg{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5004/wiki/desc/1.png)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}