iGEM 2020 — Team SZU-China

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# Team:SZU-China/Notebook

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99

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## Notebook

Notebook

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# Team:SZU-China/Engineering

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[SAFETY](https://2020.igem.org/Team%3ASZU-China/Safety)

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* Experiment
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  + [Notebook](https://2020.igem.org/Team%3ASZU-China/Notebook)

- [Models](https://2020.igem.org/Team%3ASZU-China/Model)
  * [Virtual Mutation](https://2020.igem.org/Team%3ASZU-China/Model/1)
  * [Expression model](https://2020.igem.org/Team%3ASZU-China/Model/2)
  * [Orthogonal test](https://2020.igem.org/Team%3ASZU-China/Model/3)
  * [BP-NN and GA](https://2020.igem.org/Team%3ASZU-China/Model/4)
- Human practices
  * [Integrated HP](https://2020.igem.org/Team%3ASZU-China/Human_Practices)
  * [Education](https://2020.igem.org/Team%3ASZU-China/Education)
  * [Meetups](https://2020.igem.org/Team%3ASZU-China/Meetups)
- [Safety](https://2020.igem.org/Team%3ASZU-China/Safety)
- Awards
  * [Integrated HP](https://2020.igem.org/Team%3ASZU-China/Human_Practices)
  * [Education](https://2020.igem.org/Team%3ASZU-China/Education)
  * [Models](https://2020.igem.org/Team%3ASZU-China/Model)
  * [Hardware](https://2020.igem.org/Team%3ASZU-China/Hardware)
  * [Excellent in Another Area](https://2020.igem.org/Team%3ASZU-China/Excellence_in_Another_Area)
  * [Achievements](https://2020.igem.org/Team%3ASZU-China/Achievement%20)

99

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## Engineering Success

* [Bio-indigo](#Bio)
* [Gardenia Blue](#Gardenia)
* [Cellulase Washing](#Cellulase)
* [References](#References)

![](https://static.igem.org/mediawiki/2020/5/50/T--SZU-CHINA--es1.png)

Bio-indigo

First generation of blue dye

Blue is the essence of jeans, and they all come from a long-standing dye called indigo, first extracted from plants, such as wood blue, isatis indica, indigo blue, etc. These days, jeans have been popular all over the world, and the annual output jeans have reached several billion pieces. Most of the jeans dyes are chemically synthetic indigo which brings many environmental problems. What's worse, a variety of toxic and harmful chemicals, such as hydrogen cyanide, formaldehyde, are involved in the dyeing processes. But many factories directly discharge sewage without treatment to reduce costs. That is why textile dyeing has become one of the most polluting industries in the world.

![](https://static.igem.org/mediawiki/2020/8/8e/T--SZU-CHINA--ES-1.png)

##### Fig.1 A river in Xintang, "Hometown of Jeans" (Image source: Google satellite)

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 bio-indigo.

In the blue indigo plants, such as Polygonum tinctorium, Indigofera tinctoria, Baphicacanthus cusia, and Isatis tinctoria, cells of these plants contain the secondary metabolites indican (indoxyl-β-D-glucoside) and isatan B (1H-Indol-3-yl β-D-ribo-3-hexosulopyranoside) as indigo precursors. The mechanisms of indigo production in P. tinctorium showed that indican is stored in vacuoles and is degraded to indoxyl and β-D-glucose following the release of β-D-glucosidase from chloroplasts. Subsequently, indoxyl is oxidized by air to form the dimer indigo but only under conditions of cell lysis.

SZU-China 2020 are planning to produce bio-indigo 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.

When searching in the iGEM library, we dramatically found that iGEM13\_Berkeley, iGEM19\_GreatBay\_SZ and iGEM19\_Tongji\_China have all tried to produce bio-indigo. Great minds think alike! At the same time, we contacted SHANGHAI\_SFLS\_SPBS, who also wants to produce bio-indigo this year.

We used the part of iGEM19\_GreatBay\_SZ to construct the recombinant tnaA-Fmo plasmid and express in DH5alpha.

![](https://static.igem.org/mediawiki/2020/a/a3/T--SZU-CHINA--ES-2.png)

##### Fig.2 Plasmid map of pSC101-TALEsp2-tnaA-Fmo

We used DMSO for resolubilization, and bio-indigo had an obvious absorption peak at 617nm, which was the same as that recorded in the literature. And the pigment is clearly blue in daylight.

![](https://static.igem.org/mediawiki/2020/0/07/T--SZU-CHINA--ES-3.png)

##### Fig.3 Bioidigo absorption spectrum

During the experiment, we found that the production of bio-indigo was unsatisfactory. Literature shows that if we want to increase the production of bio-indigo, we need to add a large amount of tryptophan to the culture medium. However, the cost of tryptophan far exceeds synthetic indigo, which is contrary to the original purpose of reducing costs.

![](https://static.igem.org/mediawiki/2020/1/18/T--SZU-CHINA--es9.png)

##### Fig.4 Indigo standard curve

In addition, we got the unexpected orange-red dye in the fermentation product. After we contacted with SHANGHAI\_SFLS\_SPBS, we found that this phenomenon also happened in their experiments. After consulting the literature, that may be due to the production of isatin, which is obtained by oxidation of indigo. It is preliminarily speculated that this is due to the overexpression of FMO, which caused the indigo to be further oxidized into isatin.

![](https://static.igem.org/mediawiki/2020/4/4c/T--SZU-CHINA--ES-5.png)

##### Fig.5 Unexpected orange-red dyeabsorption spectrum

Meanwhile, bio-indigo is hard to dye, for it is insoluble in water which makes it needs repeating dyeing many times to have an obvious blue colour. Therefore, we hope to develop a more environmentally friendly and lower cost blue pigment that can be more effective for denim dyeing.

Gardenia Blue

Second generation of blue dye

Due to the shortcomings of bio-indigo, we hope to find a better blue dye to replace bio-indigo. After consulting a large number of literature, we found a more environmentally friendly, low-cost blue dye, Gardenia Blue.

Gardenia Blue is a blue dye from gardenia fruits, with advantages of stable physical and chemical properties, heat resistance, acid and alkali resistance. It is easily soluble in water, making the dyeing easy.

Besides, Gardenia Blue pigment has a wildly used, because, with the addition of different amino acids, it can be shown in different colours from green, blue to purple that meets basic needs of customers for jeans.

Two-step enzymatic synthesis of Gardenia Blue wildly uses in the industry. Firstly, the extracted gardenoside transformed into genipin via the activity of β-glucosidase. Then genipin combined with primary amino acids at a high temperature, a process which generates natural blue pigments.

The one-step chemoenzymatic reaction also used in the production of Gardenia Blue, in which the enzymatic hydrolysis of geniposide and colour reaction of amino acids proceed simultaneously.

However, the colour 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.

We hope to produce β-glucosidase with high-temperature resistance. Directly adding the enzyme to the mixture of gardenoside and primary amino acids, instead of a step-by-step process, to obtain a higher quality of Gardenia Blue. It saves production costs, making it possible to obtain high-quality natural blue pigment industrially.

![](https://static.igem.org/mediawiki/2020/c/cd/T--SZU-CHINA--ES-6.png)

##### Fig.6 Blue pigment production from geniposide and amino acids

Through literature research, we found a very heat-resistant β-glucosidase (bglA) from Thermotoga Maritima, an enzyme with high solubility, high optimum reaction temperature, and good temperature stability. We hope to express bglA efficiently in E. coli, to achieve the goal of industrial production of Gardenia Blue.

To efficiently express heterologous proteins in E. coli, we optimized the bglA gene sequence for E. coli codons. And we added a GST tag to the N-terminus of bglA to improve the solubility of the protein to avoid the formation of inclusion bodies.

![](https://static.igem.org/mediawiki/2020/9/90/T--SZU-CHINA--ES-7.png)

##### Fig.7 Plasmid map of pGEX-4T-1-H-beta-glucosidase

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, and plotted the enzyme activity-temperature curve. We also tested the enzyme activity at different reaction times and plotted the enzyme activity-time curve.

![](https://static.igem.org/mediawiki/2020/7/7d/T--SZU-CHINA--es3.jpg)

##### Fig.8 a. standard curve of bata-glucosidase enzyme activity b. beta-glucosidase enzyme activity-temperature curve c. beta-glucosidase enzyme activity-time curve

The heat-resistant bglA enzyme produced by E.coli has such high activity surprised us. In the temperature of 80 degrees Celsius, it is obvious to see a blue pigment with the naked eye in five minutes reaction. The multi-salt bridge structure between the peptide chains of the thermophilic protein and the hydrogen bond on the main chain may count for its heat-resistance. To further enhance the enzyme activity, we analyzed the protein structure of bglA, found its active site, made mutations at the enzyme active site, and performed molecular docking to find mutant strains with higher enzyme activity.

After single factor analysis, we determined that our best formula is to add 440ul geniposide, 6ul glycine, 50ul enzyme to 1ml system, heating for 150 minutes in 80 seconds.

In the results, it's shown that the production of Gardenia Blue pigment using heat-resistant bglA has become very simple and efficient through the one-step chemoenzymatic reaction.

![](https://static.igem.org/mediawiki/2020/5/5d/T--SZU-CHINA--es4.png)

##### Fig.9 a. Gardenia blue pigment solution produced by different amino acids; b. OD595 of different amino acids; c. the absorption curve of gardenia blue with different amino acids

![](https://static.igem.org/mediawiki/2020/1/1b/T--SZU-CHINA--es5.png)

##### Fig.10 a. Gardenia blue yield - Geniposide Concentration curve; b. Gardenia blue yield-glycine concentration curve; c. Gardenia blue yield-temperature curve ; d. Gardenia Blue Yield-Time curve

After determined the best formula for the production of gardenia blue, we carried out mass production of gardenia blue to imitate the industrial dyeing process.

Glycine-Gardenia Blue has a better and darker coloration, which verified the superiority of glycine.

We also explored the effects of different metal mediators on dyeing. We have found that the dyeing effect is best after 40 minutes of iron pretreatment.

![](https://static.igem.org/mediawiki/2020/b/b5/T--SZU-CHINA--es6.png)

##### Fig.11 a.Influence of the dyeing time; b.the influence holding dyeing temperature; c.the influence of different metal mediators

Cellulase Washing

Fraying Process

In addition to full dyeing, there is another essential process: fraying. The traditional fraying process usually involves sand washing, water washing, sandblasting, chemical ageing 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, solsine, 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 environmental 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 do little damage to machines and fabrics.

We expect to mass-produce this enzyme in E.coli. CL34 from Streptomyces and IARI-SP-2 endo-beta-1,4-glucanase from Bacillus subtilis is endo-β-glucanases.

Those enzymes randomly cut the amorphous region inside the cellulose polysaccharide chain, destroying the entire cellulose structure, which lines on our expectations. We connect the gene to the vector to construct an expression vector.

![](https://static.igem.org/mediawiki/2020/e/e1/T--SZU-CHINA--ES-9.png)

##### Fig.12 Plasmid maps

We first characterized the CL34 enzyme activity. We drew the enzyme activity-temperature curve as follows. It's shown that the optimum temperature for CL34 is at 45°C.

As for the enzyme activity-temperature curve of IARI-SP-2 endo-beta-1,4-glucanase, the result shows that the enzyme has the highest enzyme activity at 38°C with 28.6964u. Compared with the best enzyme activity of CL34, 29.65138u, it is slightly lower by 3.22%, but it has lower temperature requirements.

The traditional fraying process does not involve heating up. Therefore, if our target enzyme has the best enzyme activity at room temperature, we can save energy as much as possible and increase economic benefits, so we chose IARI- SP-2 endo-beta-1,4-glucanase as our best enzyme.

Separately, we measured the enzyme activity-time curves of the two enzymes. It shows that not only the initial enzyme activity of CL34 reaction is much lower than IARI-SP-2 endo-beta-1,4-glucanase, but also the decay rate is much faster than IARI-SP-2 endo-beta-1,4-glucanase. It convinces us that IARI- SP-2 endo-beta-1,4-glucanase is a better choice.

![](https://static.igem.org/mediawiki/2020/1/11/T--SZU-CHINA--es8.png)

##### Fig.13 a.enzyme activity-temperature curve; b.enzyme activity-time curve

To test the actual effect of cellulase, we soaked four cloths of equal quality in IARI-SP-2 endo-beta-1,4-glucanase solution in PBS buffer. Finally, the surface was photographed with an electron microscope to observe the destruction of the cellulose structure on the surface.

In order to further improve our cellulase, we consulted Professor Liu Gang, an expert in cellulase research, and he informed us that the activity of recombinant cellulase is related to many factors. It may be that the protein is not folded correctly or the expression host is not compatible or other reasons.

However, the cellulase enzyme washing method has been used in the industry. The activity and efficiency of the enzyme are important issues. We must find a recombinant enzyme with better activity. Professor Liu Gang also suggested that we use a mixed enzyme system, which can better adapt to different working environments in the industrial system and achieve better results.

![](https://static.igem.org/mediawiki/2020/4/42/T--SZU-CHINA--poc9.png)
![](https://static.igem.org/mediawiki/2020/d/dc/T--SZU-CHINA--poc10.png)

##### Fig.14 electron micrograph of cellulase treatment

References

Shintaro Inoue, Rihito Morita, Keiko Kuwata, Tadashi Kunieda, Haruko Ueda, Ikuko Hara-Nishimura, Yoshiko Minami, Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium, Plant Physiology and Biochemistry, Volume 132, 2018, Pages 138-144, ISSN 0981-9428,https://doi.org/10.1016/j.plaphy.2018.08.034

Hsu, T., Welner, D., Russ, Z. et al. Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nat Chem Biol 14, 256–261 (2018). https://doi.org/10.1038/nchembio.2552

M. Sadeghi, H. Naderi-Manesh, M. Zarrabi, B. Ranjbar, Effective factors in thermostability of thermophilic proteins, Biophysical Chemistry, Volume 119, Issue 3, 2006, Pages 256-270, ISSN 0301-4622, https://doi.org/10.1016/j.bpc.2005.09.018.

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# Team:SZU-China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:SZU-China/Results

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[Entrepreneurship](https://2020.igem.org/Team%3ASZU-China/Entrepreneurship)
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  * [Virtual Mutation](https://2020.igem.org/Team%3ASZU-China/Model/1)
  * [Expression model](https://2020.igem.org/Team%3ASZU-China/Model/2)
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  * [Achievements](https://2020.igem.org/Team%3ASZU-China/Achievement%20)

99

![](https://static.igem.org/mediawiki/2020/c/c5/T--SZU-China--result.jpeg)

![](https://static.igem.org/mediawiki/2019/2/25/T--SZU-CHINA--left_arrow.png)

## Result

* [Plasmid Construction](#Plasmid)
* [Indigo characterization](#Indigo)
* [Beta-glucosidase](#Characterization)
* [Blue Synthetic Formula](#Best)
* [Cellulase](#Characterization_of)
* [Result](#The_result)
* [Prospect](#Prospect)
* [References](#References)

Plasmid Construction

The plasmid vector loaded with endo-glucosidase IARI-SP-2 endo-beta-1,4-glucanase gene was first synthesized and subjected to double enzyme digestion[3]. The resulting DNA electrophoresis bands are shown below. The band 1 represents the plasmid after double digestion and the band 2 represents the plasmid vector before digestion. The band M represents the reference band of KB Ladder. It can be seen that the band after digestion is about 1500bp less than before, which is in our expectation, indicating that the gene transformation is successful.

In the same way, one plasmid vector was loaded with beta-glucosidase gene[4], the other plasmid vector was loaded with CL34 cellulase gene. The former was digested with EcoRI and the latter was double digested with BamHI and NotI. After DNA electrophoresis we got the following map. The left picture shows that after double enzyme digestion, the band 1 is lower than the band 2, and the size is about 5000bp, which is the same as our prediction, indicating that the gene transformation is successful.

The right picture shows the plasmid loaded with CL34 cellulase after single enzyme digestion. M1 is a 5000bp maker, M2 is a 15000bp maker. S1 and S2 are two parallel single digestion sample sets. It can be seen that the sample sets are all in the size of about 5000bp which is the same as our plasmid vector. That indicates a successful gene transformation.

![](https://static.igem.org/mediawiki/2020/f/fc/T--SZU-CHINA--Result-1.png)

##### Fig.1 Agarose-gel electrophoresis of beta-glucosidase, IARI-SP-2, CL34

Next, we did the polyacrylamide gel electrophoresis on the three protein expressions and observed obvious protein expressions as shown in the figure.

The induced group 1 marked with a rectangular white box is our induced beta-glucosidase, and the induced II group marked with a rectangular white box is our induced IARI-SP-2 endo-beta-1,4-glucanase.

On the right, strip 1 represents the control group of untransformed plasmids, strip 2 represents the experimental group without IPTG induction, and strip 3 represents the experimental group that has been induced. Among them, the one marked by the white box is the expressed CL34.

![](https://static.igem.org/mediawiki/2020/2/27/T--SZU-CHINA--Result-2.png)

##### Fig.2 Protein electrophoresis of beta-glucosidase, IARI-SP-2, CL34

Indigo characterization

We used a part from iGEM19\_GreatBay\_SZ to construct a recombinant tnaA-Fmo plasmid and expressed it in DH5alpha. DMSO was used for resolubilization, and after scanning the absorption peak, a absorption peak is obvious at 617nm which represented the bio-indigo, as the literature expected. The resulting product solution was blue.[1]

![](https://static.igem.org/mediawiki/2020/4/41/T--SZU-CHINA--Result-3.png)

##### Fig.3 a. Indigo absorption peak spectrum. b. trial samples c.indigo standard curve. d. Bio-indigo produced by E.coil

A piece of fabric was dyed with the direct dyeing method using our bio-indigo and a standard curve was constructed for bio-indigo. The color of bio-indigo is too light and the dyeing effect is poor.

Figure c intuitively reflects the color comparison between indigo blue solution and gardenia blue solution. The sample on the left is a 0.5 mg/ml indigo blue solution, and the sample on the right is a 5 μg/ml gardenia blue solution. It is clearly shown that although the density has been different by a hundred times, the blue color of indigo is far less obvious than Gardenia Blue.

Figure d reflects the dyeing of Gardenia Blue and indigo. Sample C represents the unstained control group, Sample G represents the experimental group stained with gardenia blue, and Sample I represents the experimental group stained with biological indigo. It can be seen that the dyeing effect of indigo is much worse than that of gardenia blue.

If we need to increase the production of bio-indigo, we need to add a large amount of tryptophan to the medium. But the cost of tryptophan far exceeds the cost of synthetic indigo, which is completely contrary to our original intention of reducing costs.

![](https://static.igem.org/mediawiki/2020/f/f7/T--SZU-CHINA--Result-4.png)

##### Fig.4 a.the color contrast of indigo solution and gardenia solution b. Comparison of indigo dyeing and gardenia dyeing.

When we increase the tryptophan content in the medium, we unexpectedly got an orange-red dye in the fermentation product[2]. As noted in the literature, we found this may be due to the production of isatin dye, which was obtained by oxidation of indigo. We preliminarily speculated that it was the overexpression of FMO that caused the indigo to be further oxidized into isatin. The absorption peak spectrum of isatin obtained was also measured to further confirm its production.

![](https://static.igem.org/mediawiki/2020/b/bd/T--SZU-CHINA--Result-5.png)

##### Fig.5 a. Isatin absorption peak spectrum. b. The color contrast between bio-indigo and isatin

Characterization of beta-glucosidase

We constructed a recombinant plasmid PGEX-4T-1-H-beta-glucosidase with GST and transformed it into E.coli BL2l4. After culture in a shaker at 37°C, bacterial were collected after IPTG expression was induced, and beta-glucosidase was collected after breaking. Before the specific measurement of enzyme activity, we drew the standard curve of bata-glucosidase enzyme activity as follows.

Here, we define the unit enzyme activity per unit volume of bgla (U/ml) as the amount of 1nmol p-nitrophenol produced per unit volume per minute.

We verified its enzyme activity temperature. It can be seen that it has the best enzyme activity at 90°C, which is the same as described in the literature.

We also set a time gradient to detect the relationship between enzyme activity and reaction time at 80°C, and got the enzyme activity-time curve as shown in figure 6. It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by. The modeling part and the Gardenia Blue production-time curve also confirm this.

![](https://static.igem.org/mediawiki/2020/c/cc/T--SZU-CHINA--Result-6.png)

##### Fig.6 a. standard curve of bata-glucosidase enzyme activity. b. beta-glucosidase enzyme activity-temperature curve. c. beta-glucosidase enzyme activity-time curve.

Best Gardenia Blue Synthetic Formula

Above all, we plotted the gardenia blue standard curve as follows.

![](https://static.igem.org/mediawiki/2020/d/d6/T--SZU-CHINA--Result-7.png)

##### Fig.7 Gardenia Blue Standard Curve.

We first considered using different kinds of amino acids for the reaction. We used 20mg/ml concentration of geniposide and 0.1mol/L of the same concentration of different types of amino acids (Asp, Glu, Trp, Met, Phe, Lys , Gly) to react under the action of beta-glucosidase at 80°C for 30min. The following results were shown below.

![](https://static.igem.org/mediawiki/2020/6/68/T--SZU-CHINA--Result-8.png)

##### Fig.8 Gardenia blue pigment solution produced by different amino acids.

Intuitively, it can be seen that glycine presents an obvious blue and almost purple. Then we used the microplate reader to measure the OD595. It can be seen that glycine has the highest absorbance under 595nm light.

![](https://static.igem.org/mediawiki/2020/6/65/T--SZU-CHINA--Result-9.png)

##### Fig.9 OD595 of different amino acids.

We then scanned the product from 300nm to 700nm. There are obvious product absorption peaks near 590nm~600nm, as shown in the figure below. We noticed that the highest absorption peak of glycine at 596nm is higher than that of other amino acids. Therefore, we are more sure that glycine is the amino acid we need most.

At the same time, each amino acid has strong absorption protrusions at 300 nm, suggesting the existence of reaction intermediate products recorded in the literature, which has a strong absorption peak at 291nm.

![](https://static.igem.org/mediawiki/2020/6/65/T--SZU-CHINA--Result-10.png)

##### Fig.10 The absorption curve of gardenia blue with different amino acids.

After selecting glycine, we conducted an experiment to select the best geniposide concentration. We used geniposide as a variable, added 120μl to 680μl of geniposide solution（20mg/ml) to a 1ml reaction system, and measured the absorption peak of the solution at 595nm with a microplate reader to obtain the following figure[5].

With the increase of geniposide, the synthetic yield of Gardenia Blue gradually increases but the increase rate becomes smaller. In the group with more than 440 μl of geniposide, there was almost no significant increase in production. In the group that added less than 440 μl geniposide, the yield increased significantly. Therefore, we chose 440μl, the key dosage of geniposide, as the optimal dosage. It not only ensures high pigment production, but also avoids the waste of raw materials.

![](https://static.igem.org/mediawiki/2020/3/3a/T--SZU-CHINA--Result-11.png)

##### Fig.11 Gardenia Blue Yield- Geniposide Concentration Curve.

In the same way, we then carried out the optimal glycine concentration selection experiment[3]. We added 1μl to 17μl of glycine solution (140mg/ml）as a variable to the 1ml reaction system. After heating in a water bath at 80°C for 3 hours, we used a microplate reader to measure the absorption peak of the 595nm solution, and the following figure was obtained.

It can be seen that the dosage of 4μl glycine is the key mutation point of the curve. At this point, the system can fully convert glycine into gardenia blue pigment within 3 hours. If less than 4μl of glycine is added to the reaction system, the production of gardenia blue will be low due to lack of reactants.

With the increase in the amount of glycine, the yield increased rapidly. When more than 4μl of glycine is added, the glycine is gradually excessive but the heating temperature remains unchanged, resulting in the entire system being insufficient to completely consume the reactants within 3h.

However, due to the excess of reactants, the reaction rate is slightly greater than the reaction rate at the mutation point, so the product is still slightly improved compared to the mutation point.

Therefore, the growth rate of the production curve has dropped significantly since then. When 6μl or more of glycine is added, the increase in the reactant becomes very insignificant, and the curve reaches a plateau.

Therefore, we choose 6μl as the optimal amount of glycine, which can ensure the maximum yield of gardenia. Because the added amount of amino acids is so small that it has little effect on the cost.

![](https://static.igem.org/mediawiki/2020/2/23/T--SZU-CHINA--Result-12.png)

##### Fig.12 Gardenia blue yield-glycine concentration curve.

We then set a heating temperature gradient for gardenia blue production from 30°C to 100°C. We used 440μl of geniposide and 10μl of glycine as the reaction substrates and used a 1ml reaction system and drew the enzyme activity-temperature curve as shown in the figure.

It can be seen that the highest point of gardenia blue production lies between 80°C and 90°C. Since the output of the 90℃ group is only 1.27% higher than that of the 80℃ group, from the perspective of energy saving and economy, we chose the heating temperature of 80℃ as the best reaction temperature for our gardenia blue production.

![](https://static.igem.org/mediawiki/2020/e/e0/T--SZU-CHINA--Result-13.png)

##### Fig.13 Gardenia blue yield-temperature curve.

In order to explore the relationship between the amount of product produced and time, we used 440μl of geniposide and 10μl of amino acid as the reaction substrate at 80℃, and set up 7 groups of time gradient groups from the reaction time of 5min to 180min.

The product-time curve is shown in the figure. It can be seen that at this temperature, the reaction rate decreases significantly when the reaction proceeds to 150 minutes, and enters a plateau. This is not only confirmed by the enzyme activity-time curve, but also the production-time curve predicted in our modeling part.

In the actual situation, 150minutes is sufficient and has high economic benefits. When heated to 180min, the reactant only increases by 1.41%, which is not worth the loss for industrial production. Therefore, we chose 150min of heating time as our best reaction time.

![](https://static.igem.org/mediawiki/2020/b/b0/T--SZU-CHINA--Result-14.png)

##### Fig.14 Gardenia Blue Yield-Time Curve.

Characterization of cellulase

Before measuring the enzyme activity, we drew the standard curve of the glucose solution according to ptorocol as follows.

![](https://static.igem.org/mediawiki/2020/c/cd/T--SZU-CHINA--Result-15.png)

##### Fig.15 Glucose standard curve

Here, we define the cellulase activity concentration (U/ml) as the amount of 1μg reducing sugar produced per minute in a 1ml reaction system under the action of enzyme.

We first characterized the activity of both enzymes. We drew the enzyme activity-temperature curve as follows. It can be seen that 45℃ is the optimum temperature for CL34, the best enzyme activity is 29.65138U/ml. 38℃ is the optimum temperature for IARI-SP-2, and the best enzyme activity is 28.6964U/ml.

Compared with the best enzyme activity of CL34, although IARI-SP-2 is slightly lower by 3.22%, the temperature requirement is much lower.

The traditional fraying process does not involve heating up. Therefore, if our target enzyme has the best enzyme activity at room temperature, we can save energy as much as possible and increase economic benefits. Therefore, we chose IARI-SP-2 as an improvement.

![](https://static.igem.org/mediawiki/2020/1/1d/T--SZU-CHINA--Result-16.png)

##### Fig.16 Enzyme activity-temperature curve

Separately, we also measured the enzyme activity-time curves of the two enzymes as follows.

It can be seen that the initial enzyme activity of CL34 reaction is much lower than that of IARI-SP-2, and the decay rate is much lower than that of IARI-SP-2 much faster. Therefore, we chose IARI-SP-2 as the suitable enzyme.

![](https://static.igem.org/mediawiki/2020/a/a9/T--SZU-CHINA--Result-17.png)

##### Fig.17 Enzyme activity-time curve

The result of actual application of cellulase in distressing

We took four cloths of equal mass, soaked them in the IARI-SP-2 crude enzyme solution and added an equal volume of pH=7 PBS buffer to dilute. The quality ratio of enzyme solution and denim was controlled at 80:1[6].

We put them in a flask and placed them in a shaker at room temperature (22°C) for shaking. After 48 hours, they were taken out for inspection, and the surface was photographed with an electron microscope to observe the destruction of the cellulose structure on the surface as shown below.

The comparison shows that the fiber structure on the denim fiber bundle is basically intact before the enzyme treatment is added. After 48 hours of enzymatic treatment, the denim fiber bundle structure was obviously damaged, and significant faults and traces left by the cellulose chains were visible. The result was within our expectations.

Limited by equipment reserves, we cannot control the constant temperature and keep shaking. If a higher mass ratio is used, the purified enzyme solution is not diluted and placed in a constant temperature shaker at 38°C for reaction, the treatment effect will be more impressive.

![](https://static.igem.org/mediawiki/2020/8/87/T--SZU-CHINA--Result-18.png)
![](https://static.igem.org/mediawiki/2020/5/50/T--SZU-CHINA--Result-19.png)

##### Fig.18 Electron microscope observation of cellulose after enzymolysis

Prospect

In the future, we will add GST tags to CL34 and perform codon optimization on CL34.We will also GST-purify the GST-labeled enzymes expressed by the three genes to prepare pure enzymes and calculate specific enzyme activities.

For the preparation of gardenia blue, we will redraw the time-yield curve, reducing the gradient, and extending the time. Our ideal is to react for 24H, sampling every half an hour, a total of 48 experimental groups. At the same time, we will scan the samples of each experimental group with a wavelength of 200nm-700nm to observe the generation of the reaction intermediate products with the highest absorption peak at 291nm mentioned in the literature.

At the same time, as mentioned in the modeling section, we have made site mutations to beta-glucosidase and found several effective mutation products. We will clone these mutant genes and express the mutant enzymes, and verify their actual performance through experiments.

In addition, we plan to add a suicide system to the engineered bacteria carrying the above three enzyme genes. We will use iGEM19\_SZU-China's suicide part BBa\_K2912017, and look for a glycine controller to replace its Tryptophan attenuator (from BBa\_K2912014), and the rest remain unchanged. We hope that this controller can sense changes in the glycine concentration and turn on subsequent gene expression when the glycine concentration is higher than 1%. When we add more than 1% glycine to the bacterial solution, the R-bodies proteins that lyse the cells can be expressed. After we add glycine, the pH of the solution will just trigger the modification of R-bodies proteins, lyse the cells and release the enzymes. This will allow us to directly add the substrate for the production of gardenia blue to the bacterial solution and proceed directly to the synthesis reaction. In addition, bacteria that have not been lysed will be killed by the continuous high temperature during the production of pigments. The residue can be washed away after dyeing.

What's more, we will consider mixing CL34 and IARI-SP-2 for fraying, and then observe the damage of the surface cellulose under the electron microscope. We will also conduct a tensile test on the denim we have made to test its tensile strength.

References

[1] Wang Zhenxuan. Cloning, expression and catalytic mechanism of a flavin-dependent monooxygenase in bacteria[D].Shanghai:Shanghai Jiaotong University, 2011.

[2] Hyun-Jae Shin,Si Wouk Kim,Gui Hwan Han.Optimization of bio-indigo production by recombinant E. coli harboring fmo gene[J].Enzyme and Microbial Technology,2008,42(7):617-623.

[3] Luo Weiguang. Cloning, expression and enzymatic properties of cellulase genes derived from Bacillus subtilis[D]. Henan: Henan University of Science and Technology, 2015.

[4] Zhang Hui, Qiu Riyong, Liu Mingjie, et al. Cloning and overexpression of extremely heat-resistant β-glucosidase gene and its application in the industrial production of natural blue pigment[J].Jiangsu Agricultural Sciences,2013,41(7 ):19-22.

[5] Xu Youzhi,Liang Huazheng,Chen He, et al.Preparation and stability of high color value gardenia blue pigment[J].Modern Food Science and Technology,2011,27(4):440-443.

[6] Qiang Wang,Jiugang Yuan,Yuanyuan Yu. Cellulase immobilization onto the reversibly soluble methacrylate copolymer for denim washing[J].Carbohydrate Polymers: Scientific and Technological Aspects of Industrially Important Polysaccharides,2013,95(2):675-680.

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