Team:SZU-China/Design
Design
Indigo, the oldest and most classic pigment, gives jeans its distinctive blue color. Since indigo is insoluble in water, it cannot be used directly for dyeing. It must be reduced to soluble leucoindigo by reductant in alkaline solution and absorbs into the fibres, and then oxidized into insoluble blue indigo for dyeing.
The diffusion performance of leucoindigo is poor, and the dye liquor has a weak penetrating ability to the yarn during dyeing. Therefore, the indigo dyeing generally adopts the dyeing method of low concentration, normal temperature (or low temperature), repeated alternation of dipping and aeration.
The indigo dyeing process is complex and eco-friendly. Therefore, we hope to use engineered bacteria to produce indigo with low cost and high efficiency, which can also lay the foundation for our future production of new pigment.
Aiming at reducing the demand for the use of harmful chemicals, we are wondering if we can use synthetic biology methods to produce low-cost blue pigment.
We first tried to produce biological indigo. Bio-indigo is produced by the metabolism of tryptophan in engineered bacteria.
There are two key steps. First, we need indole, the precursor substance of indigo. Tryptophan can be transformed into indole by tryptophanase encoded by tnaA enzyme. Secondly, under the action of the flavin monooxygenase encoded by the gene FMO, the hydroxyl group is introduced into the indole to form the intermediate hydroxyindole. Finally, when the cell is lysed. Indole can spontaneously oxidize to indigo. Therefore, recombinant tryptophanase and recombinant flavin monooxygenase are expressed by engineered E.coli to achieve the purpose of biosynthesis of indigo.
Fig.1 The synthesis mechanism of indigo
We use DMSO to re-dissolve the supernatant of the disrupted cells to obtain the initial extract of biological indigo. We will perform a full-wavelength scan of the initial extract to determine the highest absorption peak wavelength and determine its color for subsequent dyeing and comparison operations.
A standard curve for the yield of bio-indigo was established, and calculate the yield of bio-indigo. Finally, we will directly dye a piece of cloth with our bio-indigo to verify its dyeing effect.
In the practical application of bio-indigo, we found that although it has the advantages of environmental friendliness, its water solubility is still worrying and there are problems with production efficiency and dyeing ability. So we found a new pigment, Gardenia Blue.
Gardenia Blue, a blue pigment from gardenia fruits, has the advantages of stable physical and chemical properties, heat resistance, acid and alkali resistance and safty.
It is now being widely used for dyeing food, medicine and cosmetics. It has good water solubility and affinity for cotton fiber, easy usage in dyeing process, and its dyeing effect is superior to indigo.
Geniposide, a substrate for the synthesis of gardenia blue, a natural substance derived from gardenia, is almost harmless to the environment. And there are mature methods and supporting systems to extract geniposide. In large-scale industrial dyeing applications, the cost of obtaining geniposide is meager.
Besides, the synthetic pathway of gardenia blue has a distinctive feature which is of promising application. That is, with the addition of different amino acids, we can obtain pigments with different colors form green, blue to purple that caters to diverse demands of customers for jeans.
Two-step enzymatic synthesis of Gardenia Blue is wildly used in the industry. Firstly, the extracted geniposide is hydrolyzed into genipin via the catalytic action of beta-glucosidase. Then genipin combined with primary amino acids at a high temperature, a process that generates natural blue pigments.
The one-step chemoenzymatic reaction also used in the production of Gardenia Blue, in which the enzymatic hydrolysis of geniposide is followed by the color reaction of amino acids automatically in the same reaction vessel.
However, the color reaction carried out at a high temperature, where most industrial β-glucosidase can not tolerate, which makes it hard to implement such a simplified method.
Those methods involve a key enzyme: β-glucosidase. It belongs to the cellulase system, which can hydrolyze the non-reducing β-D-glycosidic bond bound to the terminal, and release β-D-glucose and the corresponding ligand at the same time. In the synthetic pathway of gardenia blue, one molecule of glucose is removed form geniposide due to the hydrolyzation of beta-glucosidase. Thus, geniposide was converted into genipin. After that, genipin will reacts with amino compounds like amino acid to produce gardenia blue. And different amino acids as reactant can generate pigments with different colors.
Fig.2 Synthesis mechanism of gardenia blue
We need to find an enzyme that has good tolerance to extreme industrial environments, which can also achieve rapid color reaction in large quantities.
We finally chose a heat-resistant β-glucosidase from Thermus marinus as our experimental enzyme.
We can directly add the heat-resistant β-glucosidase to the mixture of geniposide and primary amino acid to obtain higher quality gardenia blue pigment at high temperatures. This is a further improvement to the two-step method for producing Gardenia Blue, which cut the costs and makes it possible to obtain high-quality natural blue pigment.
We chose the plasmid pGEX-4T-1-H and added a GST tag to it, which facilitated the subsequent purification of GST and obtained pure protein samples and helped improve the solubility of the protein and avoid the formation of inclusion bodies. This system ensures that, by adding IPTG induction, genes can be efficiently expressed to produce the enzymes we need under laboratory conditions. In addition, since the original host bacteria of this enzyme is not Escherichia coli, we have optimized its gene sequence for E. coli codons. The optimized gene can be more suitable for the expression system of E. coli, laying the foundation for efficient expression.
Fig.3 Construction of β-glucosidase plasmid vector
We first digested the transformed bacteria with plasmid restriction enzymes and ran DNA electrophoresis to confirm that the transformation was successful. Then, we disrupted the cells after inducing, extracted the supernatant protein solution, and ran SDS-PAGE to verify the successful expression of our protein.
After that, we tested the enzyme activity of β-glucosidase, and set a temperature gradient to find the temperature at which it reached the maximum enzyme activity. We also tested the enzyme activity at different reaction times and plotted the enzyme activity-time curve.
After confirming the successful characterization of β-glucosidase, we set out to explore the production conditions of Gardenia Blue.
As mentioned earlier, the production of gardenia blue involves two reactants: geniposide and amino acids. Since the different amino acid types will significantly affect their color, we set up an experimental group with different amino acids.
After determining the best amino acid we chose, we carried out controlled variable experiments with different addition amounts of the same amino acid and different addition amounts of geniposide as variable respectively. After determining the best formula, we explored the most economical reaction time for the production of geniposide.
For the mass-production of Gardenia Blue, we strive to mimic the dyeing process in the industry. We used the direct dyeing method and the post-mordant dyeing method to dye the white denim. We also set up different bath ratio gradients for the two dyeing methods to explore the best dyeing conditions.
In addition, according to our reference, we thought that mordant dyeing would lead to a better color fastness by adding metal ion fixatives.And metal ions are widely used in dyeing factories nowadays. However, there is no significant difference in color fastness of our dyed fabrics regardless of whether the metal ions were added.This shows that our pigment does not need the assistance of metal ions to improve color fastness.This once again reflects our advantages of environmental friendliness and high dyeing effectiveness.
Fig.4 Schematic diagram of dyeing process
As for a piece of clothing after dyeing, it is inevitable to withstand conditions such as washing, soaping, and perspiration. We have designed corresponding testing experiments for these situations. We will work with local testing agencies to complete these tests.
In addition to full dyeing, there is another essential process: fraying. The traditional fraying process usually involves sand washing, water washing, sandblasting, chemical aging and other steps. With the low cost of those processes, many small jeans factories wildly accepted those traditional fraying process. However, it consumes a lot of water, accompanied by the use of a large amount of toxic and harmful chemical reagents such as indigo, alkali and potassium manganate, which not only does harm to the health of workers in the jeans factory but also the environment. The remaining problems hurt the health of consumers who buy such jeans. The current environmentally friendly fraying process, the laser, is too expensive for most factories.
The purpose of traditional methods such as sand washing is to destroy the structure of the cellulose on the surface of the jeans to achieve a worn-out effect. Therefore, we hope to adopt a more eco-friendly and low-cost method to complete the process of fraying.
We hope to use cellulase to achieve this effect. The cellulase hydrolyzes the cellulose, causing a part of the indigo dye to fall off the fabric to achieve the "worn feeling." Relevant literature points out that compared with stone washing, cellulase washing can obtain the same effect as stone washing. Besides, cellulase washing has unique advantages such as high efficiency, non-toxicity and harmlessness, and little damage to machines and fabrics.
We finally chose two genes, the IARI-SP-2 endoglucanase from Bacillus subtilis and the CL34 endoglucanase from Streptomyces. Those enzyme cuts the amorphous region inside the cellulose polysaccharide chain, produces oligosaccharides of different lengths and new chain ends, thus breaks down the entire cellulose structure, which is in line with our expectations. We loaded CL34 into pet-28a vector, and IARI-SP-2 into pGEX-4T-1-H vector. For IARI-SP-2, we have carried out codon optimization to optimize its expression in E.coli. with the GST tag.
Fig.5 Cellulase plasmid construction
We detected the enzyme activity of CL34 and IARI-SP-2, set the temperature gradient, and drew the enzyme activity-temperature curve. After that, we also tested the enzyme activity at different reaction times and drew the enzyme activity-time curve to determine the most suitable enzyme for us.
In addition, by simulating the method of cellulase treatment in the industry, we add the fabric to the washing machine to co-react with cellulase. After each treatment, we observe the damage of the cellulose layer on the surface under the electron microscope.
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