Difference between revisions of "Part:BBa K314012"
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===Usage and Biology===
 
===Usage and Biology===
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To test BBa _K314012, it was inserted into a pET29b+ vector using [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD 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.
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To test BBa _K314012, it was inserted into a pET29b+ vector using [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel 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.
  
 
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[[image:Kinetic_CapDCP.png|1600px|thumb|center| Kinetic constants, ''k''cat and Km, taken from our plotted curves are shown in the table above.]]
 
[[image:Kinetic_CapDCP.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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[[Image:CapDCP_MassSpecNoMethionine.png|400px|left|thumb|''' We also ran the purified protein samples through a mass spectrometer to make sure the first methionine was being removed by the natural ''E. coli'' methionine aminopeptidase and that the protein was the expected size.  Our results indicate that a massive majority of the protein in our purified sample was within .02% error of the predicted CapD_CP size. Expected weight of CapD_CP without Methionine=55285Da, with Methionine=55417Da. Our mass spec detected a peak at 55274.8Da (no Methionine) well within the 0.02% error limit for our mass spec''']]
 
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[[Image:CapDCP_MassSpecNoMethionine.png|600px|thumb|center| We also ran the purified protein samples through a mass spectrometer to make sure the first methionine was being removed by the natural ''E. coli'' methionine aminopeptidase and that the protein was the expected size. Our results indicate that a massive majority of the protein in our purified sample was within .02% error of the predicted CapD_CP size. Expected weight of CapD_CP without Methionine=55285Da, with Methionine=55417Da. Our mass spec detected a peak at 55274.8Da (no Methionine) well within the 0.02% error limit for our mass spec]]
  
 
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Latest revision as of 20:43, 27 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 [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel 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.

  • The protein gel above shows the single band seen CapD_CP. This makes quantifying active CapD_CP extremely easy when correcting for enzyme concentration.

Kinetic constants, kcat and Km, taken from our plotted curves are shown in the table above.


We also ran the purified protein samples through a mass spectrometer to make sure the first methionine was being removed by the natural E. coli methionine aminopeptidase and that the protein was the expected size. Our results indicate that a massive majority of the protein in our purified sample was within .02% error of the predicted CapD_CP size. Expected weight of CapD_CP without Methionine=55285Da, with Methionine=55417Da. Our mass spec detected a peak at 55274.8Da (no Methionine) well within the 0.02% error limit for our mass spec


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]