Difference between revisions of "Part:BBa K391007"
(→Characterization Data) |
(→Characterization Data) |
||
| Line 3: | Line 3: | ||
== Characterization Data == | == Characterization Data == | ||
| − | The activity of DspB was tested by taking a crude cell lysate from <i>E.coli</i> cells expressing the protein. The protein in the lysate was then tested against a chromogenic substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide. As DspB cleaves this substrate, the absorbance shifts from 300nm (substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide) to 405nm (product: 4-nitrophenol | + | The activity of DspB was tested by taking a crude cell lysate from <i>E.coli</i> cells expressing the protein. The protein in the lysate was then tested against a chromogenic substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide. As DspB cleaves this substrate, the absorbance shifts from 300nm (substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide) to 405nm (product: 4-nitrophenol). We measured the change in the level of absorbance at 405nm over time and the result is shown below. |
<div style="text-align:center;">[[Image:Igem2010_assay2_linegraph.jpg|500px]]</div> | <div style="text-align:center;">[[Image:Igem2010_assay2_linegraph.jpg|500px]]</div> | ||
The graph above plots the absorbance at wavelength 405nm (y-axis) over time in days (x-axis). It can be observed from the graph that over time, the absorbance at 405nm increases with the treatment with the lysate containing DspB by a significant amount compared to the controls. This assay has been replicated and the results have shown to be reproducible. The absorbance values are fairly low for the DspB treatment because the protein was added to the substrate as a part of the crude cell lysate, not as a purified protein. Further characterization using purified DspB is recommended for results with higher absorbance at 405nm in a shorter period of time. | The graph above plots the absorbance at wavelength 405nm (y-axis) over time in days (x-axis). It can be observed from the graph that over time, the absorbance at 405nm increases with the treatment with the lysate containing DspB by a significant amount compared to the controls. This assay has been replicated and the results have shown to be reproducible. The absorbance values are fairly low for the DspB treatment because the protein was added to the substrate as a part of the crude cell lysate, not as a purified protein. Further characterization using purified DspB is recommended for results with higher absorbance at 405nm in a shorter period of time. | ||
Latest revision as of 01:00, 28 October 2010
Constitutive promoter-RBS-DspB
Strongest constitutive promoter in J23100 family (J23100) + strongest RBS in the community collection (B0034)+ Dispersin B carbohydrate digesting enzyme (K391006).
Characterization Data
The activity of DspB was tested by taking a crude cell lysate from E.coli cells expressing the protein. The protein in the lysate was then tested against a chromogenic substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide. As DspB cleaves this substrate, the absorbance shifts from 300nm (substrate: 4-Nitrophenyl N-acetyl-β-D-glucosaminide) to 405nm (product: 4-nitrophenol). We measured the change in the level of absorbance at 405nm over time and the result is shown below.
The graph above plots the absorbance at wavelength 405nm (y-axis) over time in days (x-axis). It can be observed from the graph that over time, the absorbance at 405nm increases with the treatment with the lysate containing DspB by a significant amount compared to the controls. This assay has been replicated and the results have shown to be reproducible. The absorbance values are fairly low for the DspB treatment because the protein was added to the substrate as a part of the crude cell lysate, not as a purified protein. Further characterization using purified DspB is recommended for results with higher absorbance at 405nm in a shorter period of time.