Part:BBa_K515107:Experience
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how you used this part and how it worked out.
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.
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
A time-lapse video (below) 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.
A video of another photoconversion of Dendra in E. coli cells that have been taken up into Arabidopsis roots can be seen in the video below.
Our project is still in the very early stages of development. It will need to go through several levels of safety testing and optimisation before it can be implemented (Figure 1).
INSERT FIGUREPredicted developmental stages of the AuxIn project.
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