Difference between revisions of "Part:BBa K3360002"
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===SZU-China 2020 TEAM=== | ===SZU-China 2020 TEAM=== | ||
| − | Beta-glucosidase is a cellulase enzyme, which can catalyze the reaction of geniposide with amino acids to produce gardenia blue pigment. In order to find an enzyme that has | + | Beta-glucosidase is a cellulase enzyme, which can catalyze the reaction of geniposide with amino acids to produce gardenia blue pigment. In order to find an enzyme that has good tolerance to the extreme industrial environment and can achieve rapid color development in laboratory conditions, we finally created an extremely heat-resistant β-glucosidase derived from <i>Thermotoga marina</i>. |
| − | We chose the plasmid pGEX-4T-1-H, and added a GST tag to it and optimized its gene sequence for E. coli codons. Under IPTG induction, the enzyme can be expressed efficiently. | + | We chose the plasmid pGEX-4T-1-H, and added a GST tag to it and optimized its gene sequence for <i>E. coli</i> codons. Under IPTG induction, the enzyme can be expressed efficiently. |
[[File: beta-glucosidase Plasmid vector diagram.png|500px|center]] | [[File: beta-glucosidase Plasmid vector diagram.png|500px|center]] | ||
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<center> Fig.2 Agarose-gel electrophoresis of beta-glucosidase</center> | <center> Fig.2 Agarose-gel electrophoresis of beta-glucosidase</center> | ||
| − | Next, we did the polyacrylamide gel electrophoresis on | + | Next, we did the polyacrylamide gel electrophoresis on these 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. |
[[File: Protein electrophoresis of beta-glucosidase.png|200px|center]] | [[File: Protein electrophoresis of beta-glucosidase.png|200px|center]] | ||
<center> Fig.3 Protein electrophoresis of beta-glucosidase</center> | <center> Fig.3 Protein electrophoresis of beta-glucosidase</center> | ||
| − | We constructed a recombinant plasmid pGEX-4T-1-H-beta-glucosidase with GST and transformed it into E.coli BL2l. 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 beta-glucosidase enzyme activity . | + | We constructed a recombinant plasmid pGEX-4T-1-H-beta-glucosidase with GST and transformed it into <i>E.coli</i> BL2l. 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 beta-glucosidase enzyme activity. |
Here, we define the unit enzyme activity per unit volume of beta-glucosidase (U/ml) as the amount of 1nmol p-nitrophenol produced per unit volume per minute. | Here, we define the unit enzyme activity per unit volume of beta-glucosidase (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 | + | 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℃, and got the enzyme activity-time curve as shown in the figure. It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by. |
| − | [[File: standard curve of bata-glucosidase enzyme activity.png| | + | [[File: standard curve of bata-glucosidase enzyme activity.png|900px|center]] |
| − | <center> Fig.4 | + | <center> Fig.4 （a) standard curve of bata-glucosidase enzyme activity (b) beta-glucosidase enzyme activity-temperature curve (c) beta-glucosidase enzyme activity-time curve</center> |
| − | + | ||
| − | + | ||
| + | Then we follow our best formula: add 440μl geniposide, 6μl glycine, 50μl beta-glucosidase enzyme solution, and 504μl PBS buffer to the 1ml reaction system. And we prepared 250ml gardenia blue pigment solution. The color and output are within our expectations. | ||
| + | [[File: Homemade Gardenia Blue Solution.jpeg|500px|center]] | ||
| + | <center> Fig.5 Homemade Gardenia Blue Solution</center> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
Latest revision as of 18:05, 27 October 2020
Thermostable beta-glucosidase
Encoding the β-glucosidase A of Thermotoga maritima, the entire sequence has been optimized by E.coli codons, and the N-terminus is marked with GST to increase its solubility and reduce the formation of inclusion bodies.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1577
Illegal AgeI site found at 927 - 1000COMPATIBLE WITH RFC[1000]
SZU-China 2020 TEAM
Beta-glucosidase is a cellulase enzyme, which can catalyze the reaction of geniposide with amino acids to produce gardenia blue pigment. In order to find an enzyme that has good tolerance to the extreme industrial environment and can achieve rapid color development in laboratory conditions, we finally created an extremely heat-resistant β-glucosidase derived from Thermotoga marina.
We chose the plasmid pGEX-4T-1-H, and added a GST tag to it and optimized its gene sequence for E. coli codons. Under IPTG induction, the enzyme can be expressed efficiently.
We synthesized the above-mentioned vector and double-enzyme digested it. After DNA electrophoresis, the following map was obtained. The 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.
Next, we did the polyacrylamide gel electrophoresis on these 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.
We constructed a recombinant plasmid pGEX-4T-1-H-beta-glucosidase with GST and transformed it into E.coli BL2l. 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 beta-glucosidase enzyme activity.
Here, we define the unit enzyme activity per unit volume of beta-glucosidase (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℃, and got the enzyme activity-time curve as shown in the figure. It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by.
Then we follow our best formula: add 440μl geniposide, 6μl glycine, 50μl beta-glucosidase enzyme solution, and 504μl PBS buffer to the 1ml reaction system. And we prepared 250ml gardenia blue pigment solution. The color and output are within our expectations.
