Difference between revisions of "Part:BBa E0040"
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====Purificaton==== | ====Purificaton==== | ||
| − | GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http:// | + | GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.6 protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.6 results]for the purification can be found. |
====Calibration==== | ====Calibration==== | ||
| − | The fluorescence of purified GFPmut3B was calibrated in the [[Chassis/Cell-Free_Systems|cell-free chassis]]. The derived [http:// | + | The fluorescence of purified GFPmut3B was calibrated in the [[Chassis/Cell-Free_Systems|cell-free chassis]]. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page. |
[[Image:E0040_invitro_calibration.png|thumb|center|500px|Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.]] | [[Image:E0040_invitro_calibration.png|thumb|center|500px|Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.]] | ||
====Degradation==== | ====Degradation==== | ||
| − | The degradation of GFPmut3B in the [[Chassis/Cell-Free_Systems|cell-free chassis]] was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http:// | + | The degradation of GFPmut3B in the [[Chassis/Cell-Free_Systems|cell-free chassis]] was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.4 Detailed protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.4 results]are attached. |
[[Image:E0040 invitro degradation.png|thumb|left|500px|Degradation curve of different GFP concentrations.]] | [[Image:E0040 invitro degradation.png|thumb|left|500px|Degradation curve of different GFP concentrations.]] | ||
[[Image:E0040 invitro deglog.png|thumb|none|500px|Semi-log plot of GFP degradation at different GFP concentrations]]<br clear="all"> | [[Image:E0040 invitro deglog.png|thumb|none|500px|Semi-log plot of GFP degradation at different GFP concentrations]]<br clear="all"> | ||
From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis. | From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis. | ||
| + | ====Improvements==== | ||
| + | Yeast- and FACS optimized GFP: <html><a href="https://parts.igem.org/Part:BBa_K194001" target="_blank">BBa_K194001</a> | ||
| + | Yeast- and FACS optimized, fast degradable GFP: <html><a href="https://parts.igem.org/Part:BBa_K194002" target="_blank">BBa_K194002</a> | ||
Revision as of 02:59, 22 October 2009
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Usage and Biology
Untagged version of gfp from Repressilator reporter. See the design page for more source information.
Fluorescence wavelengths
Cormack et al.Cormack report the following excitation and emission data for GFPmut3 -
- Excitation max - 501nm
- Emission max - 511nm
Latency
Cormack et al.Cormack report detectable fluorescence within 8 mins. Please add maturation time data for E0040 here.
References
<biblio>
- Cormack pmid=10659856
</biblio>
Part Characteristics in Cell-Free Chassis
| Parameter | Value and Description |
|---|---|
| Calibration | A conversion factor of 79.429 from Au to concentraion in nM |
| Half-life | 33 hours in the cell-free chassis, with a degradation constant of 0.0210 (in hours) |
Purificaton
GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.6 protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.6 results]for the purification can be found.
Calibration
The fluorescence of purified GFPmut3B was calibrated in the cell-free chassis. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page.
Degradation
The degradation of GFPmut3B in the cell-free chassis was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.4 Detailed protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.4 results]are attached.
From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis.
Improvements
Yeast- and FACS optimized GFP: BBa_K194001 Yeast- and FACS optimized, fast degradable GFP: BBa_K194002