Part:BBa_E0040

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.

The original citation for GFPmut3b is as follows:

Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996).

Here's the link: http://www.sciencedirect.com/science/article/pii/0378111995006850

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>

1. 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.

Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.

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.

Degradation curve of different GFP concentrations.

Semi-log plot of GFP degradation at different GFP concentrations

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