Difference between revisions of "Part:BBa K314012"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | To test BBa _K314012, it was inserted into a pET29b+ vector using Kunkel's Mutagenesis. CapD_CP was then produced and purified as described in the [http://2010.igem.org/Team:Washington/Protocols/50mLPurificationCapD UW 2010 iGEM Team's Small Scale Protein Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki]. A protein gel was also run to determine physical qualities | + | To test BBa _K314012, it was inserted into a pET29b+ vector using Kunkel's Mutagenesis. CapD_CP was then produced and purified as described in the [http://2010.igem.org/Team:Washington/Protocols/50mLPurificationCapD UW 2010 iGEM Team's Small Scale Protein Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki]. A protein gel was also run to determine physical qualities. The resulting data is shown below. |
<gallery heights=300px widths=400> | <gallery heights=300px widths=400> | ||
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The substrate vs. reaction rate curve above plots the PDG cleaved per enzyme per hour (y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, high substrate concentrations suffered from substrate inhibition under the conditions our enzyme was assayed. Kinetic constants, ''k''cat and Km, taken from our plotted curves are shown in the table above. | The substrate vs. reaction rate curve above plots the PDG cleaved per enzyme per hour (y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, high substrate concentrations suffered from substrate inhibition under the conditions our enzyme was assayed. Kinetic constants, ''k''cat and Km, taken from our plotted curves are shown in the table above. | ||
| − | The protein gel above shows the single band seen CapD_CP. This makes CapD_CP extremely easy | + | The protein gel above shows the single band seen CapD_CP. This makes quantifying active CapD_CP extremely easy when correcting for enzyme concentration. |
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 18:11, 22 October 2010
CapD_CP
A circularly permuted [http://www.parts.igem.org/Part:BBa_K314011 CapD] with a Foldit-designed linker.
Usage and Biology
To test BBa _K314012, it was inserted into a pET29b+ vector using Kunkel's Mutagenesis. CapD_CP was then produced and purified as described in the [http://2010.igem.org/Team:Washington/Protocols/50mLPurificationCapD UW 2010 iGEM Team's Small Scale Protein Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki]. A protein gel was also run to determine physical qualities. The resulting data is shown below.
The substrate vs. reaction rate curve above plots the PDG cleaved per enzyme per hour (y-axis) as a function of substrate concentration (x-axis). As observed in the curve above, high substrate concentrations suffered from substrate inhibition under the conditions our enzyme was assayed. Kinetic constants, kcat and Km, taken from our plotted curves are shown in the table above.
The protein gel above shows the single band seen CapD_CP. This makes quantifying active CapD_CP extremely easy when correcting for enzyme concentration. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1489
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]