Difference between revisions of "Part:BBa K590023"
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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. | 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| | + | [[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.]] |
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| + | {| {{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> | ||
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| + | | [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> | ||
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| + | | [https://parts.igem.org/wiki/index.php?title=Part:BBa_K590021 '''wt-Kumamolisin''']||(3.25 ± 0.05) x 10<sup>4</sup> | ||
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| + | | SC-PEP ||(4.96 ± 0.75) x 10<sup>3</sup> | ||
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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.
| Mutation(s) | kcat/KM (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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1696
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]