Part:BBa_K515107:Experience
Characterisation
This part (BBa_K515107) has been characterised in a number of aspects to test its properties as a reporter. The tests describe this part in terms of thermostability, photostability and photoconversion.
Thermostability
This test is to show the thermostability of Dendra, by finding the temperature at which the protein denatures. Stock solutions of Dendra were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.
After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The samples were then measured by fluorescence on a 96-well plate. The corresponding curve was plotted on a graph
As can be seen from the graph, Dendra 2 denatures at 88°C. This makes it very useful for both in vivo and in vitro reporting as it can withstand high temperatures.
Photostability
This test is to show photostability of Dendra protein, for green fluorescence without the conversion to red fluorescence. Green (505nm wavelength) and red (575nm wavelength) fluorescence emission was measured for Dendra expressing cells over time. As control RFP and GFP expressing cells have been used to compare the green and red fluorescence emission. Red and green fluorescence of empty LB medium was also measured as a blank that can be subtracted from the readings of the GFP, RFP and Dendra expressing cells.
Photoconversion
Purified Dendra protein was measured for the red fluorescence emission after photoconversion using single photon stimulation at 405nm wavelength. It was exposed to the RFP excitation wavelength (558nm) and at a set time point it was also exposed to 405nm photoconversion wavelength.
Figure 1: Red flourescence emission of Dendra protein upon single photon stimulation at 405nm wavelength. Red flourescence is very low before photoconversion, however at time point 25s after the start of the measurement 405nm photoconverting wavelength was applied. An increase in red flourescence emission can be observed between time point 25s and 319s, after which red flourescence emission levels off.
This part has been used as a reporter for observation of bacterial uptake into the roots of the plants. Due to its photoconvertible properties, it allows monitoring of the metabolic activity of bacterial cell once uptaken into the root. Dendra was converted from 486nm excitation and 505nm emission wavelength, to 558nm excitation and 575nm emission wavelength using single photon stimulation. Conversion was achieved after exposure to 405nm wavelength using laser. Photoconversion was completed after about 15 rounds of bleaching at 50% laser intensity with the pinhole set to 3 airy units.
Figure 5. Dendra2 photoconversion in bacteria taken up inside plant roots. The graph on the top displays averaged fluorescence over the entire photoconverted area and the amount of brightfield light (the background light used to see the outline of the roots) recorded is therefore very high. The graph on the bottom displays emission at green and red fluorescence over the same time span. |
Video 3. This video shows the photoconversion of Dendra2 within E. coli cells that have been taken up into the plant roots as a time-lapse of pictures taken after each round of bleaching at 405nm. The targeted area of cells being photoconverted corresponds to the top graph in Figure x. There is a single bacterium visible on the right that was not targeted for photoconversion and serves as a control (data and imaging by Imperial College iGEM 2011). |
Intensity ROI 1
After exposure with 405nm wavelength, the cells are observed to steadily decrease fluorescing in green emission spectra over a time period of 140 seconds. In the same time frame the cells are observed to increase fluorescing in the red spectra, with fluorescence in the two emission spectra being equally intense at 20 seconds after the photoconversion. Brightfield emmision is kept at just over 100 units throughout the duration of observation of photoconversion. Brightfield is present for visualisation of the root and bacterial cells without flourescence.
Intensity ROI 2
ROI 2 displays the fluorescence emitted by a single cell while it is being converted from green to red emission. Emission of green spectra fluorescence can be observed to decrease steadily over time period of 140 seconds. In the same time frame the cell can be observed to fluoresce more strongly in the red spectra. The difference between red and green emission is greater in ROI 2 than in ROI 1 due to single cell focus eliminating backround emission which can be seen in ROI 1 that causes the difference between two spectra in ROI 1 to be smaller than in ROI 2. Brightfield emmision is kept at just over 100 units throughout the duration of observation of photoconversion. This is necessary for visual observation of cells within the root.
A single bacterium on the right (visible in the video above) is used as a negative control. It was not targeted for bleaching by the laser and can be observed to remain green through the conversion process.
Method
The bacterial cells within root were visualised using a Zeiss LSM-510 inverted confocal microscope. The induction of photoconversion was performed using a laser at 405nm wavelength. The bacterial root uptake experiment used the same protocol as previously described by Paungfoo-Lonhienne et al. (1)References:
(1) Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7): e11915. http://www.nih.gov/science/models/arabidopsis/index.html
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