Difference between revisions of "Part:BBa K314015"
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<partinfo>BBa_K314015 short</partinfo>
 
<partinfo>BBa_K314015 short</partinfo>
  
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A mutated CapDCP protein aimed at increasing hydrolysis and decreasing transpeptidation.
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A mutated [http://www.parts.igem.org/Part:BBa_K314012 CapD_CP] protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG.
  
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To test BBa _K314015, it was inserted into a pET29b+ vector using Kunkel's Mutagenesis. CapD 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 against CapD_CP, for a detailed description of the assay please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki].  The resulting data is shown below.
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===Usage and Biology===
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To test BBa _K314015, it was inserted into a pET29b+ vector using [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel Mutagenesis]. CapD_CP-F24H 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 against CapD_CP. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki].  The resulting data is shown below.
  
 
<gallery heights=400px widths=350>
 
<gallery heights=400px widths=350>
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image:CapD_MM.png
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image:F24H_MM.png|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.
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image:F24H_MM.png
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</gallery>
 
</gallery>
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[[image:Kinetic_F24H.png|1600px|thumb|center| Kinetic constants, ''k''cat and Km, taken from our plotted curves are shown in the table above.]]
  
 
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Latest revision as of 20:43, 27 October 2010

CapD_CP-F24H

A mutated [http://www.parts.igem.org/Part:BBa_K314012 CapD_CP] protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG.

Usage and Biology

To test BBa _K314015, it was inserted into a pET29b+ vector using [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel Mutagenesis]. CapD_CP-F24H 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 against CapD_CP. For a detailed description of the assay, please see the [http://2010.igem.org/Team:Washington/Protocols/EnzymeAssayCapD 2010 UW iGEM wiki]. 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1489
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]