Difference between revisions of "Part:BBa K590023"

Revision as of 00:01, 29 October 2011

Kumamolisin-As_N291D

A mutated Kumamolisin-As enzyme aimed to break down gluten by increased activity with the PQLP peptide, an antigenic epitope in gliadin.

Usage and Biology

This part was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease. To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. Kumamolisin-As_N291D 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. Error bars represent a 95% confidence interval from triplicate data.

Mutation(s)	k_cat/K_M (M^-1 s^-1)
N291D	(5.21 ± 0.11) x 10⁵
S354N, D358G, D368H	(2.45 ± 0.02) x 10⁵
G319S, D358G, D368H	(2.02 ± 0.02) x 10⁵
G319S, D358G, D368H, N291D (KumaMax)	(3.86 ± 0.04) x 10⁶
wt-Kumamolisin	(3.25 ± 0.05) x 10⁴
SC-PEP	(4.96 ± 0.75) x 10³

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]
Unknown
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 This part was constructed by the [http://2011.igem.org/Team:Washington 2011 University of Washington] iGEM team to break down gluten, the primary cause of Celiac's disease.   To test BBa _K590021, it was inserted into a protein expression vector, pET29b+. Kumamolisin-As_N291D 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_N291D.png|750px|center|thumb|The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.]]
+[[Image:Washington_N291D.png|500px|left|thumb|The relative activity plot was produced by measuring the quantity of PQLP peptide cleaved per enzyme per hour. Error bars represent a 95% confidence interval from triplicate data.]]
+{| {{table}}
+| align="center" style="background:#f0f0f0;"|'''Mutation(s)'''
+| align="center" style="background:#f0f0f0;"|'''''k<sub>cat</sub>''/''K<sub>M</sub>'' (M<sup>-1</sup> s<sup>-1</sup>)'''
+|-
+| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590023 '''N291D''']||(5.21 ± 0.11) x 10<sup>5</sup>
+|-
+| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590024 '''S354N, D358G, D368H''']||(2.45 ± 0.02) x 10<sup>5</sup>
+|-
+| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590022 '''G319S, D358G, D368H''']||(2.02 ± 0.02) x 10<sup>5</sup>
+|-
+| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590087 '''G319S, D358G, D368H, N291D (KumaMax)''']||(3.86 ± 0.04) x 10<sup>6</sup>
+|-
+| [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||(3.25 ± 0.05) x 10<sup>4</sup>
+|-
+| SC-PEP ||(4.96 ± 0.75) x 10<sup>3</sup>
+|-
+|}
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