Team:SDU-Denmark/project-p

From 2010.igem.org

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(UV-Vis 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 in both 2D and 3D, while also tracking them over time.
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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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2.5 ul of the bacterial dilution were placed on the center of the microscopy slide and covered by a coverslide. 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 previously experiment, with the exeption that instead of optical filters we used diodes that emitted light at the wanted wavelengths (500nm and 600nm respectively). Moreover we also recorded the bacteria's behaviour when exposed to a blue light gradient and varying intensities of light measured in milliampere. All the measurements were carried out on the modified phototactic bacteria and the wildtype MG1655.<br>
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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>
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The results look like these:<br>
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The resulting videos looked like these examples:<br>
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<object width="320"><param name="movie" value="http://www.youtube.com/v/IlPRLSEG864?hl=de&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/IlPRLSEG864?hl=de&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="320"></embed></object> <object width="320"><param name="movie" value="http://www.youtube.com/v/HC6DRAWcHys?hl=de&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/HC6DRAWcHys?hl=de&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="320"></embed></object><br>
<object width="320"><param name="movie" value="http://www.youtube.com/v/IlPRLSEG864?hl=de&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/IlPRLSEG864?hl=de&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="320"></embed></object> <object width="320"><param name="movie" value="http://www.youtube.com/v/HC6DRAWcHys?hl=de&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/HC6DRAWcHys?hl=de&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="320"></embed></object><br>
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The bacteria that are not moving in the video are assumed to be sticking to the glass or coverslip.  
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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.<br>
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Here is an example of a .txt file containing the results: [http://2010.igem.org/wiki/images/6/6b/TrackResult_iGEM-WTred1.txt].
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=== Stability assay ===
=== Stability assay ===
To determine the stability of our pSB1C3-K343007 plasmid, a stability experiment was carried out according to protocol[[http://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343007 was grown in LB media without chloramphenicol, whereby no selection pressure is exerted on the bacteria. Dilutions of the culture was spread on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloramphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generations.<br><br>
To determine the stability of our pSB1C3-K343007 plasmid, a stability experiment was carried out according to protocol[[http://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343007 was grown in LB media without chloramphenicol, whereby no selection pressure is exerted on the bacteria. Dilutions of the culture was spread on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloramphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generations.<br><br>

Revision as of 16:01, 27 October 2010