Team:SZU-China/Results
Result
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.
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.
Fig.2 Protein electrophoresis of beta-glucosidase, IARI-SP-2, CL34
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]
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.
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.
Fig.5 a. Isatin absorption peak spectrum. b. The color contrast between bio-indigo and isatin
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.
Fig.6 a. standard curve of bata-glucosidase enzyme activity. b. beta-glucosidase enzyme activity-temperature curve. c. beta-glucosidase enzyme activity-time curve.
Above all, we plotted the gardenia blue standard curve as follows.
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.
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.
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.
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.
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.
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.
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.
Fig.14 Gardenia Blue Yield-Time Curve.
Before measuring the enzyme activity, we drew the standard curve of the glucose solution according to ptorocol as follows.
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.
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.
Fig.17 Enzyme activity-time curve
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.
Fig.18 Electron microscope observation of cellulose after enzymolysis
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.
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