Team:SDU-Denmark/project-p

From 2010.igem.org

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'''1. Growth of bacterial culture on semi-solid agar plates (Experiment 1):'''<br>
'''1. Growth of bacterial culture on semi-solid agar plates (Experiment 1):'''<br>
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The bacteria and plates were prepared after our own protocol, which you can find here: [http://2010.igem.org/Team:SDU-Denmark/protocols#Photosensor_characterisation] <br>
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The bacteria and plates were prepared after our own protocol, which you can find here: [http://2010.igem.org/Team:SDU-Denmark/protocols#Photosensor_characterisation] <br> [[Image:Ingeniør1.JPG|200px|thumb|left|Wildtype bacteria]][[Image:Ingeniør4.JPG|200px|thumb|left|Phototactic bacteria]]
Since exposure to blue light should decrease the phototaxic bacterias tumbling frequency, the expected result was that the colony which was placed between the light and dark half of the plate would spread out in the darkness and would not move further when it reached the light. This is counter-intuitive, since decreased tumbling should lead to a longer distance traveled. What happens at the microscopic scale in semisolid agar is that the agar creates a matrix like structure where there are channels through the agar, which the bacteria can swim through. The decrease in tumbling frequency of the bacteria will make it harder for them to find the channels in the agar to swim through, which leads to them being trapped where they were placed. The result is that a colony which shows an increased run time, will look as if it it was non-motile on these plates. Our results showed exactly this; the bacterial culture had spread out to on the dark half of the plate and did not get nearly as far on the half exposed to light. This experiment was done with a normal wildtype MG1655 and a non-motile strain of ''E.coli'', DH5alpha, as controls. As expected these cells did not show anything like the behavior described above, which indicates that the effect stems from the modification to our photosensor bacteria. These results were useable, but not fully conclusive, since there were some non-optimal conditions present in this experiment. We used ambient light instead of pure blue light, and the exposure to light for the multiple samples was not exactly even. Therefore we had to improve our experiment setup and see if we could reproduce these results with a more reliable setup.<br>
Since exposure to blue light should decrease the phototaxic bacterias tumbling frequency, the expected result was that the colony which was placed between the light and dark half of the plate would spread out in the darkness and would not move further when it reached the light. This is counter-intuitive, since decreased tumbling should lead to a longer distance traveled. What happens at the microscopic scale in semisolid agar is that the agar creates a matrix like structure where there are channels through the agar, which the bacteria can swim through. The decrease in tumbling frequency of the bacteria will make it harder for them to find the channels in the agar to swim through, which leads to them being trapped where they were placed. The result is that a colony which shows an increased run time, will look as if it it was non-motile on these plates. Our results showed exactly this; the bacterial culture had spread out to on the dark half of the plate and did not get nearly as far on the half exposed to light. This experiment was done with a normal wildtype MG1655 and a non-motile strain of ''E.coli'', DH5alpha, as controls. As expected these cells did not show anything like the behavior described above, which indicates that the effect stems from the modification to our photosensor bacteria. These results were useable, but not fully conclusive, since there were some non-optimal conditions present in this experiment. We used ambient light instead of pure blue light, and the exposure to light for the multiple samples was not exactly even. Therefore we had to improve our experiment setup and see if we could reproduce these results with a more reliable setup.<br>
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[[Image:Ingeniør1.JPG|200px|thumb|right]][[Image:Ingeniør4.JPG|200px|thumb|right]]
 
'''2. Growth of bacterial culture on semi-solid agar plates (Experiment 2):'''<br>
'''2. Growth of bacterial culture on semi-solid agar plates (Experiment 2):'''<br>
The experiment described above was repeated in a more controlled environment. This means that there were no changes as to how the plates or bacterial cultures were prepared, so we refer again to [http://2010.igem.org/Team:SDU-Denmark/protocols#Photosensor_characterisation] in regards to how this was done. The difference lies in the setup of the light-controlled environment. What we did this time was that the plates was illuminated from above by a single light source in an otherwise completely dark environment, we prepared a cut-out so that the light would only hit one half of our plates and the other half would remain in the dark. [[Image:Lightbox5k.gif|thumb|Schematic over plate positioning and areas exposed to light.]] Since the problem with the last experiment was that the light source was just normal white light (which contains a lot of different wavelengths), this time around we used an optical filter so that only light with a wavelength of around 470nm could pass through, which resulted in a blue light shining down on the plates. The light-source itself was a run-of-the-mill flashlight, with a blue light filter installed in front of the lens. To eliminate the effect of temperature gradients inside the incubator, the three samples were placed in a triangle formation in the center of the incubator. <br>
The experiment described above was repeated in a more controlled environment. This means that there were no changes as to how the plates or bacterial cultures were prepared, so we refer again to [http://2010.igem.org/Team:SDU-Denmark/protocols#Photosensor_characterisation] in regards to how this was done. The difference lies in the setup of the light-controlled environment. What we did this time was that the plates was illuminated from above by a single light source in an otherwise completely dark environment, we prepared a cut-out so that the light would only hit one half of our plates and the other half would remain in the dark. [[Image:Lightbox5k.gif|thumb|Schematic over plate positioning and areas exposed to light.]] Since the problem with the last experiment was that the light source was just normal white light (which contains a lot of different wavelengths), this time around we used an optical filter so that only light with a wavelength of around 470nm could pass through, which resulted in a blue light shining down on the plates. The light-source itself was a run-of-the-mill flashlight, with a blue light filter installed in front of the lens. To eliminate the effect of temperature gradients inside the incubator, the three samples were placed in a triangle formation in the center of the incubator. <br>

Revision as of 14:21, 24 October 2010