Difference between revisions of "Part:BBa E0040"

Revision as of 03:28, 28 October 2017

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

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.

Expression of GFP-mut3b in cell lysate

Cell-free GFP-mut3b synthesis was analyzed in self-made E.Coli lysate from strain BL21(DE3). Fluorescence was measured at 37°C for five hours on a plate reader. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our [http://2017.igem.org/Team:EPFL/Protocols protocols]. Shown are three repeats with a negative control as well as a shaded error graph (control was subtracted) summarizing the result. GFP-mut3b expression yields high signals in lysate and thus is a good choice of reporter while working on a cell-free chassis. Saturation occurs after about five hours.

Three repeats and control of GFP-mut3b expression in cell lysate.

Shaded error graph of GFP-mut3b expression in cell lysate (control was subtracted).

IIT Delhi 2017 - Characterization of Photobleaching

Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.

Here, we characterize the photobleaching effect in wildtype GFP (E0040) using fluorescent microscopy with the etaluma Lumascope 500 microscope. Cells expressing GFP under the PhlF repressible promoter (BBa_K2525016) in the absence of PhlF, so that it constitutively expressed GFP. Cells were loaded in microfluidic chambers and droplet encapsulation was performed to capture a small number of cells. This droplet was continuously exposed to light corresponding to the excitation wavelength of GFP (~485 nm) and the emission was captured continuously as well. The real time video for photobleaching in the cells encapsulated in the droplet is shown in GIF 1. ImageJ was used to analyze the images to obtain the rate of photobleaching as shown in Fig 1. Where we have fitted an exponential curve to the total intensity over time. It is known that photobleaching has a first order decay. We obtain a photobleaching rate of 0.002 per second (7.2 per hour).

GIF. 1 Real time video showing photobleaching in cells encapsulated in a droplet. To access the video in the GIF you'll have to visit the GIF at its link on the wiki; for that, click twice over the image in succession.

Fig. 1 Total Intensity in the encapsulated droplet over time. Since photobleaching is known to be a first order process, we have fitted an exponential curve to the data. The high R-squared value implies a good fit of the exponential model to the data. The rate of photobleaching turns out to be 0.002 per second (7.2 per hour).

Improvements

• Chlamydomonas reinhardtii chloroplast optimised: BBa_K2148009
• Yeast- and FACS optimized GFP: BBa_K194001
• Yeast- and FACS optimized, fast degradable GFP: BBa_K194002
• RFC[1000] compatible GFP: BBa_K2294444