Team:SDU-Denmark/project-p

From 2010.igem.org

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(HPLC determination of beta-carotene and retinal production)
(Computerized analysis of the bacterial motility with the THOR prototype by Unisensor A/S and the Unify software)
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===Computerized analysis of the bacterial motility with the THOR prototype by [http://unisensor.dk/ Unisensor A/S] and the Unify software===
===Computerized analysis of the bacterial motility with the THOR prototype by [http://unisensor.dk/ Unisensor A/S] and the Unify software===
For this experiment we changed our protocol for cultivating swimming bacteria in order to optimize their mobility [http://2010.igem.org/Team:SDU-Denmark/protocols#PS1.2 PS1.2]<br>
For this experiment we changed our protocol for cultivating swimming bacteria in order to optimize their mobility [http://2010.igem.org/Team:SDU-Denmark/protocols#PS1.2 PS1.2]<br>
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The machine and software used for the analysis are prototypes and still under heavy development. Because of trade secrets it is impossible for us to explain how the machine works or give a detailed explanation of it's mechanism until the THOR has reached production status. Simplistically said it is a very advanced video microscope, with which it is possible to analyse liquids and the particles inside those in both 2D and 3D, while also tracking them over time and also space, since the camera/optical lens can also move along the slide, while recording.<br>
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The machine and software used for the analysis are prototypes and still under heavy development. Because of trade secrets it is impossible for us to explain how the machine works or give a detailed explanation of it's mechanism until the THOR has reached production status. Simplistically said it is a very advanced video microscope, with which it is possible to analyse liquids and the particles inside those in both 2D and 3D, while also tracking them over time and also space, since the camera/optical lens can also move along the slide, while recording. <br>
2.5 ul of the bacterial dilution were placed on the center of the microscopy slide and covered by a cover slip, which results in a layer of liquid with a height of 6 um. Afterwards the sample was sealed with mineral oil as to prevent a flow in the liquid. The data collection was done exactly as described in the previous experiment, with the exception that we diodes instead of optical filters. The light used for recording was a red LED diode with a wavelength of 660 nm. The lightsource used for the exposure was a bluelight LED, which emitted light at a wavelength of 470 nm, which hit the sample with an angle of attack of 17°. Moreover we also recorded the bacteria's behaviour when exposed to a blue light gradient and varying intensities of light measured in milliampere ranging from 1 mA to 20 mA. All the measurements were carried out on the modified phototactic bacteria and the wildtype MG1655 as a control.<br>
2.5 ul of the bacterial dilution were placed on the center of the microscopy slide and covered by a cover slip, which results in a layer of liquid with a height of 6 um. Afterwards the sample was sealed with mineral oil as to prevent a flow in the liquid. The data collection was done exactly as described in the previous experiment, with the exception that we diodes instead of optical filters. The light used for recording was a red LED diode with a wavelength of 660 nm. The lightsource used for the exposure was a bluelight LED, which emitted light at a wavelength of 470 nm, which hit the sample with an angle of attack of 17°. Moreover we also recorded the bacteria's behaviour when exposed to a blue light gradient and varying intensities of light measured in milliampere ranging from 1 mA to 20 mA. All the measurements were carried out on the modified phototactic bacteria and the wildtype MG1655 as a control.<br>
The resulting videos looked like these examples:<br>
The resulting videos looked like these examples:<br>
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<br></html>
<br></html>
The bacteria that are not moving in the video are assumed to be sticking to the glass or coverslip.<br>
The bacteria that are not moving in the video are assumed to be sticking to the glass or coverslip.<br>
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From these videos we extracted data through Unisensor's in-house programmed software Unify and it's tracking extension. For each frame the bacteria were identified and their location compared to that of the frame before it and by thta, tracking the bacteria over time. The resulting data that was output for each frame was: Location of bacteria given as coordinates (X,Y), bacterial orientation in angle ranging from -180° to 180° and length of the path was tracked in pixels.<br>
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From these videos we extracted data through Unisensor's in-house programmed software Unify and it's tracking extension. For each frame the bacteria were identified and their location compared to that of the frame before it and by thta, tracking the bacteria over time. The resulting data that was output for each frame was: Location of bacteria given as coordinates (X,Y), bacterial orientation in angle ranging from -180° to 180° and length of the path was tracked in pixels. An example of the unify sofware analysing the paths of the bacteria and at the same creating a video of the individual tracks:<br>
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<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/lARwMFCay_Q?hl=de&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/lARwMFCay_Q?hl=de&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object></html><br>
Here is an example of a .txt file containing the results: [http://2010.igem.org/wiki/images/6/6b/TrackResult_iGEM-WTred1.txt Tracks of wildtype bacteria exposed to red light]. If interested, please request the whole dataset from us, which will be sent out as a 450mb zip file.<br>
Here is an example of a .txt file containing the results: [http://2010.igem.org/wiki/images/6/6b/TrackResult_iGEM-WTred1.txt Tracks of wildtype bacteria exposed to red light]. If interested, please request the whole dataset from us, which will be sent out as a 450mb zip file.<br>
From this file, a trackplot was generated, showing each bacteria's path over the recorded time:<br>
From this file, a trackplot was generated, showing each bacteria's path over the recorded time:<br>

Revision as of 18:58, 27 October 2010