Difference between revisions of "Part:BBa K3360004"
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We chose the CL34 endoglucanase from Streptomyces. We loaded CL34 into pet-28a vector as shown below. Under the induction of IPTG, CL34 will be expressed in large quantities. | We chose the CL34 endoglucanase from Streptomyces. We loaded CL34 into pet-28a vector as shown below. Under the induction of IPTG, CL34 will be expressed in large quantities. | ||
| + | [[File:Plasmid vector diagram.png|200px|center]] | ||
| + | <center>Fig1. Plasmid vector diagram</center> | ||
| + | We synthesized the above-mentioned vector and digested it with EcoRI. After DNA electrophoresis, the following map was obtained. The 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 successful gene transformation. | ||
| + | |||
| + | [[File:Agarose-gel electrophoresis of CL34.png|200px|center]] | ||
| + | <center>Fig.2 Agarose-gel electrophoresis of CL34</center> | ||
| + | |||
| + | Next, we did the polyacrylamide gel electrophoresis on the this protein expressions and observed obvious protein expressions as shown in the figure. 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. | ||
| + | |||
| + | [[File:Protein electrophoresis of CL34.png|400px|center]] | ||
| + | <center>Fig.3 Protein electrophoresis of CL34</center> | ||
| + | |||
| + | We constructed a recombinant plasmid pET-28a-CL34 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 . | ||
| + | |||
| + | [[File: Glucose standard curve.png|400px|center]] | ||
| + | <center>Fig.4 Glucose standard curve</center> | ||
| + | |||
| + | 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 characterized the activity of CL34. We drew the enzyme activity-temperature curve as follows. It can be seen that 45℃ is the optimum temperature for CL34. The enzyme activity reached 29.65138U/ml. We also measured the enzyme activity-time curves of CL34 as follows.It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by. | ||
| + | |||
| + | [[File: CL34 enzyme activity-temperature curve.png|400px|center]] | ||
| + | <center>Fig.5 CL34 enzyme activity-temperature curve | ||
| + | </center> | ||
| + | [[File: CL34 enzyme activity-time curve.png|400px|center]] | ||
| + | <center>Fig.6 CL34 enzyme activity-time curve</center> | ||
Revision as of 21:35, 23 October 2020
endo-beta-1_4-glucanase from Streptomyces
Encoding the endo-beta-1,4-glucanase of Streptomyces.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 431
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
SZU-China 2020 TEAM
The traditional fraying process is very harmful to the environment, resources and users. In essence, being old is the destruction of cellulose on the surface of jeans. We finally consider using cellulase to achieve this effect. Through cellulase washing, it can hydrolyze the cellulose and make a part of the dye fall off the fabric. Through this effect, the "worn feeling" effect that people want can be obtained.
We chose the CL34 endoglucanase from Streptomyces. We loaded CL34 into pet-28a vector as shown below. Under the induction of IPTG, CL34 will be expressed in large quantities.
We synthesized the above-mentioned vector and digested it with EcoRI. After DNA electrophoresis, the following map was obtained. The 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 successful gene transformation.
Next, we did the polyacrylamide gel electrophoresis on the this protein expressions and observed obvious protein expressions as shown in the figure. 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.
We constructed a recombinant plasmid pET-28a-CL34 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 .
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 characterized the activity of CL34. We drew the enzyme activity-temperature curve as follows. It can be seen that 45℃ is the optimum temperature for CL34. The enzyme activity reached 29.65138U/ml. We also measured the enzyme activity-time curves of CL34 as follows.It can be seen that the enzymatic reaction rate becomes slower and slower as time flows by.
