Designed by: Jiacheng Shi   Group: iGEM21_HUST-China   (2021-10-15)

The synthesis and decolorization system of indigo


As a composite part, it consists of Panb1-α factor-FMO-AOX1 Terminator-Pynr071c-α factor-Laccase-AOX1 Terminator- Pynr071c-ROX1-AOX1 Terminator. Panb1, as a constitutive promoter, will express FMO under any circumstances and secret them out of the cell through the signal peptide, α-factor. When the substrate, indole, is added, it reacts in order and catalyze indole into indigo. Pynr071c promoter, as a xylose-inducible promoter, translates and expresses laccase in the presence of xylose. Laccase oxidizes indigo to colorless substances. At the same time, it also induces the expression of ROX1. ROX1 inhibits the Panb1 promoter and blocks the expression of its downstream proteins, thus blocking the synthesis of pigment.

Usage and Biology


Flavin-containing monooxygenase, FMO is one kind of microsomal enzyme widely found in ER of most tissue, whose activity relies on flavin adenine dinucleotide (FAD), reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen. FMO could catalyze the oxidation of most exogenous compounds containing Nitrogen, Sulphur, Phosphorus, Selenium and other nucleophilic elements, which is a vital detoxifying process of carcinogen and many other hazardous substances. FMO is a kind of monooxygenase, which could produce C4a peroxyflavin intermediate, which is highly stable due to spectrum observation and could remain unchanged under 4℃ for minutes, even hours. In the first cycle, NADPH reduces FAD into FADH2. This reduced flavin could react with oxygen rapidly and become peroxyflavin, which is the main form of FMO and further react wih an appropriate nucleophilic substrate. This reaction will form one molecular of water and transfer one oxygen atom to the substrate. The release of NADP+ may be the rate-limiting step of catalytic cycle while the Vmax of substrate binding has little impact on both reactions.


Copper atom is usually contained in the laccase, which is one of the common characters of laccase and maintains its oxidizing/reducing reaction. As the key element of reaction center, copper atom is critical to the activity and specificity of laccase since it exists in the area that binds substrate and breaks & forms chemical bonds. According to crystal structure of laccase, most have 3 copper binding sites which bind 4 copper atoms. Laccase has a wide variation of substrates, which includes phenol, aniline, carboxylic acid (and their derivatives), bio-pigments, lignans, organic metal compounds and other non-phenol compounds. The optimum temperature of laccase is relatively low, while it has the maximum catalyzing efficient among environment of low pH. These characters give laccase a promising prospect in food industry, paper industry, textile industry and bio-repairing of soil. Coriolus versicolor from Aphyllophorales, Aphyllophorales, Hymenomycetes, Basidiomycota, is one of the ideal strains which is used for laccase production and have excellent degradation ability to lignans. Laccase in plants is involved in various botanic metabolism pathways, such as synthesis of lignans, synthesis and degradation of pigments and elongation of roots.

Background related to curcumin

The most ancient pigment known to humanity, indigo, now is popular in food, medic and dyeing industries. The pigment application of indigo could date back to at least 2,500 BC. and found on some blue hemp fabrics excavated from the Chinese MaWangDui and Egyptian mummies. One branch of Chinese Yao nationality is named after indigo as LanDianYao due to its unique technology of indigo dyeing. Among the food industry, indigo is used as edible pigment in the form of sodium sulfonate or aluminum, known as "bright blue" and bright blue aluminum lake in China, while being used mainly in its sodium sulfonate in the United States, called as "Indigo element" .

Hair dyeing experiment

We measured the standard curves of three pigments before using them for hair dyeing experiment. We also found that the amount of melanin contained in hair can have a significant effect on hair dyeing outcomes. Therefore, we define different colors of hair based on bleaching.


We have gained the best dye conditions of three kinds of hair dye(indigo, curcumin and lycopene) at a certain concentration. Under optimal conditions, we dyed 4-9 degrees of hair to get a series of dyeing discs. And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.

Dye/Condition time temperature Dyeing aid ingredients concentration(g/L) comment
indigo 2min Room temperature none 2 The color deepens significantly while dyeing for multiple times

Under the best conditions, we dyed the hair from 4 degree to 9 degree, and got a series of colors. It is found that it only needed to be bleached to 8 degree so that the hair would show a bright color for all three kinds of dye. As to indigo hair, 7 to 9 degree hair would become blue. As the dyeing time goes, the color would turn blue from an indigo color; 5 to 6 degree hair would be dyed to celadon, and 4 degree hair was still brown.

The dyeing results of indigo (room temperature,2g/L). From left to right: 9°(0.5,2,6min),8°(0.5,2,6min), 7°(0.5,2,6min),6°(0.5,2,6min), 5°(0.5,2,6min),4°(0.5,2,6min)

Indigo: Difficult: we can make indigo paste, but the hair does not dye well. Solution: Indigo is a water-soluble component, and need to be oxidized to indigo after fixing to the hair. With water-in-oil paste as the matrix, indigo white can not fully enter the interior of the hair, and the oily substances in the matrix and excessive reductant prevent indigo white from oxidation in the hair, resulting in no effective coloring. So we decided to design a timely manner in which indigo could be produced and used at the same time. Therefore, we consider that indigo dye can be produced and used in time -- the direct production of indigo by yeast, and the production of indigo solution as a dye in time. For this idea, we dye indigo solution directly on the hair and find that it can be painted, but it can not color the hair evenly. So we designed a hair dye comb to make it possible to evenly smear indigo cryptosomes on the hair. The matching device is a timely fermentation tank, which can meet the needs of users with our engineering bacteria as raw materials, timely production and timely use of indigo white. For detailed information, please refer to Hardware part.


Color fastness is an important aspect to measure the effect of dye, so we design a set of elution scheme and test the color fastness of three kinds of natural pigment dye products and the same color traditional dye paste. The results showed that the color fastness of the natural pigment dyes was better than that of the traditional dyes.


Sequence and Features

Assembly Compatibility:
  • 10
  • 12
    Illegal NheI site found at 4272
    Illegal NotI site found at 8106
  • 21
    Illegal XhoI site found at 124
    Illegal XhoI site found at 649
    Illegal XhoI site found at 3774
  • 23
  • 25
    Illegal NgoMIV site found at 706
    Illegal AgeI site found at 1925
    Illegal AgeI site found at 4689
  • 1000
    Illegal BsaI site found at 1928
    Illegal BsaI site found at 7025
    Illegal SapI site found at 7284