Difference between revisions of "Part:BBa K1131002:Experience"
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Revision as of 04:58, 27 September 2013
This experience page is provided so that any user may enter their experience using this part.
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how you used this part and how it worked out.
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 circled.
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