Coding
G319S_Tri

Part:BBa_K590022

Designed by: Sydney Gordon, Daniel Hadidi, Elizabeth Stanley, Sarah Wolf, Angus Toland, Sean Wu   Group: iGEM11_Washington   (2011-09-15)
Revision as of 05:29, 16 September 2011 by Wujsean (Talk | contribs) (→‎Usage and Biology)

Kumamolisin-As_G319S, D358G, D368H

A mutated Kumamolisin-As enzyme aimed to combat celiac disease by increased specificity for the PQLP peptide, an antigenic epitope in gliadin.

Usage and Biology

To test BBa _K590022, it was inserted into a pET29b+ vector using [http://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]. Kumamolisin-As was then produced and purified as described in the [http://2011.igem.org/Team:Washington/Protocols/50mL_UW 2011 iGEM Team's Small Scale Protein Expression and Purification Protocol]. The purified protein was then tested for activity. For a detailed description of the assay, please see the [http://2011.igem.org/Team:Washington/Protocols/Purified_Enzyme_Assay 2011 UW iGEM Purified Enzyme Assay Protocol]. The resulting data is shown below.

The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour.

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 1696
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 447
    Illegal NgoMIV site found at 720
    Illegal NgoMIV site found at 1248
    Illegal NgoMIV site found at 1494
    Illegal AgeI site found at 772
    Illegal AgeI site found at 1348
    Illegal AgeI site found at 1588
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None