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
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.
Photoconversion using confocal microscope
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 1: On the left, pictures of bacteria expressing BBa_K515107. 1 is the area photoconverted using the 405nm laser. 2 is an individual bacterium whose Dendra protein has undergone photoconversion. 3 is a negative control consisting of a non-photoconverted bacterium. On the right, graph representation of the photoconversion in the 3 marked areas. Intensity ROI 1 corresponds to area 1, Intensity ROI 2 corresponds to area 2 which is a single photoconverted cell, Intensity ROI 3 corresponds to area 3, which a single non-converted cell. The area which was focused on in ROI 3 is larger than in ROI 2 and therefore flourescence is much lower, this results from selection of backround area as well as the single cell. Three different emmision spectra were observed, Ch2: emission in green spectrum. Ch3: emission in red spectrum. ChD: brightfield emission.
Video 1:A time-lapse video shows the conversion of cells in area 1. The single cell in area 3 serves as a negative control. It was not bleached by the laser and therefore continued to absorb light at a lower wavelength and emit green fluorescence.
Video 2:A video of another photoconversion of Dendra in E. coli cells that have been taken up into Arabidopsis roots can be seen.
Intensity ROI 1
After exposure with 405nm wavelength, the cells in the region 1 are observed to decrease green spectra emission steadily over time period of 140 seconds. In the same timeframe the cells are observed to increase red spectra emission steadily, with the two emission spectra having the same flourescence 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
Single cell has been converted red spectra emission to after exposure with 405nm wavelength single photon stimulation. Emission of green spectra can be observed to decrease steadily over time period of 140 seconds. In the same time frame cell is observed to increase red spectra emission steadily. The emission spectra are same at a time point between 20s and 40s. The same emission of the two spectra can be measured to just over 100 units. 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 root.
Intensity ROI 3
Negative control of bacterial cell within root that has not been converted by single photon stimulation to red emission spectrum. Green spectra emission can be observed to be slightly higher than the red spectra emission. The small difference between the two spectra can be atributed to backround emission resulting from the measured spectra emission in the blue circle (3) on the right with only one measured cell within this area. Brightfield emission is kept at just over 100 units for visulisation same as in the ROI 1 and ROI 2.
Method
The bacterial cells within root have been visualised using Zeiss LSM-510 inverted confocal microscope. The induction of photoconversion was performed using laser at 405nm wavelength. The bacterial root uptake experiment have 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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