Difference between revisions of "Part:BBa K1131002:Experience"
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[[File:glupurificationconstruct.png|400px]] [[File:glugel.png]] <br> | [[File:glupurificationconstruct.png|400px]] [[File:glugel.png]] <br> | ||
| − | We then applied Michaelis-Menten kinetics to measure the activity of our glucosidase with indican and two common substrates X-gal and ONPG whose structures are depicted. | + | We then applied Michaelis-Menten kinetics to measure the activity of our glucosidase with indican and two common substrates X-gal and ONPG whose structures are depicted (X-gal on the left and ONPG on the right). |
[[File:xgal.png]][[File:onpg.png | 250px]] <br> | [[File:xgal.png]][[File:onpg.png | 250px]] <br> | ||
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Here is a time course in which we measure the increasing product formation for each of these enzymes as a function of time. We use this data to also plot the rate of reaction vs time to obtain the Km and Vmax of glucosidase with each substrate. The data is shown below. <br> | Here is a time course in which we measure the increasing product formation for each of these enzymes as a function of time. We use this data to also plot the rate of reaction vs time to obtain the Km and Vmax of glucosidase with each substrate. The data is shown below. <br> | ||
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[[File:xgaldatapage.png | 500px]] <br> | [[File:xgaldatapage.png | 500px]] <br> | ||
[[File:onpgdatapgae.png | 500px]] <br> | [[File:onpgdatapgae.png | 500px]] <br> | ||
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| + | == Citations == | ||
| + | [http://www.uniprot.org/uniprot/Q03506 Uniprot Link to Sequence] | ||
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| + | Paavilainen, S. "Purification, Characterization, Gene-Cloning, and Sequencing of a new-beta glucosidase from Bacillus Circulans subsp. alkaophilus." Applied and Environmental Microbiology. 59.3 (1993): 927-932. Print. | ||
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| + | Paavilainen, Sari. "The CrystalStructure of B-glucosidase from Bacillus Circulans sp. alkalophilus: Ability to form long polymeric assemblies." Journal of Structural Biology. 129. (2000): 69-79. Print. | ||
===User Reviews=== | ===User Reviews=== | ||
Latest revision as of 18:46, 27 September 2013
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Applications of BBa_K1131002
This B-glucosidase from Bacillus circulans can hydrolyze the B-glycosidic linkage of indican, a solubilized precursor to indigo, as well as other common reporter compounds such as ONPG and X-Gal. The reaction we were most concerned with, the cleavage of indican, is depicted below:
We proceeded to characterize the enzyme and its activity with the three substrates previously mentioned: indican, X-gal, and ONPG. For this data, we can see that this glucosidase can be used as an alternative to LacZ galactosidase in X-Gal and ONPG assays.
Proof of Functionality
We obtained basic colorimetric proof that our enzyme functions by adding 50mM indican to glucosidase in E. Coli cell lysate and observing a rapid color change. The construct we used is below:
We characterized the rate of activity our glucosidase has on each of the named substrates using standard Michaelis-Menten kinetics. From this, we can see that our glucosidase can be used as a potential alternative to LacZ galactosidase in ONPG and X-Gal colorimetric assays.
Kinetics
To observe the kinetic activity of our glucosidase, we used the T7 expression system, his6 tagged gene, and BL21 chemically competent cells to overexpress and purify this B-glucosidase. Given that this protein comes from a bacterial source, we did not forsee any problems in purification which was indeed the case. Below is the plasmid used to express this glucosidase as well as the resulting protein gel with the eluted protein fraction indicated.
We then applied Michaelis-Menten kinetics to measure the activity of our glucosidase with indican and two common substrates X-gal and ONPG whose structures are depicted (X-gal on the left and ONPG on the right).
Here is a time course in which we measure the increasing product formation for each of these enzymes as a function of time. We use this data to also plot the rate of reaction vs time to obtain the Km and Vmax of glucosidase with each substrate. The data is shown below.
Citations
[http://www.uniprot.org/uniprot/Q03506 Uniprot Link to Sequence]
Paavilainen, S. "Purification, Characterization, Gene-Cloning, and Sequencing of a new-beta glucosidase from Bacillus Circulans subsp. alkaophilus." Applied and Environmental Microbiology. 59.3 (1993): 927-932. Print.
Paavilainen, Sari. "The CrystalStructure of B-glucosidase from Bacillus Circulans sp. alkalophilus: Ability to form long polymeric assemblies." Journal of Structural Biology. 129. (2000): 69-79. Print.
User Reviews
UNIQ897dcf677bc97298-partinfo-00000000-QINU UNIQ897dcf677bc97298-partinfo-00000001-QINU