Difference between revisions of "Part:BBa K515107:Experience"

Revision as of 23:52, 14 September 2011

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Applications of BBa_K515107

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

Photostability

Photoconversion

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 to monitor the metabolic activity of the 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 flourescence 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.