Difference between revisions of "Part:BBa K590022"

Revision as of 19:33, 16 September 2011

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_G319S, D358G, D368H 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]

@@ Line 5: / Line 5: @@
 ===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.
+To test BBa _K590022, it was inserted into a pET29b+ vector using [http://2011.igem.org/Team:Washington/Protocols/Kunkel Kunkel Mutagenesis]. Kumamolisin-As_G319S, D358G, D368H 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.
 [[Image:Washington_G319S,_D358G,_D368H.png|750px|center|thumb|The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour.]]