Difference between revisions of "Part:BBa K314011"

Revision as of 21:53, 26 October 2010

CapD

A protein native to Bacillus anthracis which cleaves poly-D-gamma-glutamate (PDG) 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'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]. 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.

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

Sequence and Features