Part:BBa_K314011

CapD

A protein native to Bacillus anthracis which cleaves poly-γ-D-glutamate (PDGA) and anchors it to the bacterium's peptidoglycan.

Usage and Biology

To test BBa _K314011, we first inserted it into a pET29b+ vector using [http://2010.igem.org/Team:Washington/Protocols/KunkelCapD Kunkel 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]. A protein gel was also run to determine physical qualities. The resulting data is shown below.

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  The substrate vs. reaction rate curve above plots the Poly-d-gamma-glutamate (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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  Protein gel data above shows the three bands seen for CapD. This makes quantifying active CapD extremely difficult when correcting for protein concentration.

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 1540
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]