Team:SDU-Denmark/K343006

From 2010.igem.org

(Difference between revisions)
(K343005)
(UV-Vis determination of beta-carotene and retinal production)
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== K343005 ==  
== K343005 ==  
===UV-Vis determination of beta-carotene and retinal production===
===UV-Vis determination of beta-carotene and retinal production===
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Seeing as both beta-carotene and retinal have unique and characteristic spectres when studied by UV-vis spectrometry. The spectra can be obtained by harvesting beta-carotene- and retinal-producing cells, re-suspending them in acetone and lysing them, which we chose to do by sonication. Afterwards, the cell debris can be pelleted and the supernatant can be examinated. <br>
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Both beta-carotene and retinal have unique and characteristic spectres when studied by UV-vis spectrometry. The spectra can be obtained by harvesting beta-carotene- and retinal-producing cells, resuspending them in acetone and lysing them, which we chose to do by sonication. Afterwards, the cell debris can be pelleted and the supernatant can be examinated. <br>
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The acetone suspension of beta-carotene and retinal can then be subjected to spectrophotometry, and the obtained values and spectra can be compared to those of pure beta-carotene or retinal in known concentrations. This method provides both a qualitative answer to whether or not the desired compound is present and a qualitative indication of the concentration in the cells. <br>
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The acetone suspension of beta-carotene and retinal can then be subjected to spectrophotometry, and the obtained values and spectra can be compared to those of pure beta-carotene or retinal of known concentrations. This method provides both a qualitative answer to whether or not the desired compound is present and a quantitative indication of the concentration in the cells. <br>
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In this experiment cells were prepared and harvested according to protocol [http://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1 EX1.1]. This experiment was performed with six different strains of ''E. coli'':<br> <br>
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In this experiment, cells were prepared and harvested according to protocol [http://2010.igem.org/Team:SDU-Denmark/protocols#EX1.1 EX1.1]. This experiment was performed with six different strains of ''E. coli'':<br> <br>
Wild type TOP10<br>  
Wild type TOP10<br>  
Wild type MG1655<br>  
Wild type MG1655<br>  
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MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)<br>
MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)<br>
Both K343005 and K274210 were constitutively expressed.<br><br>
Both K343005 and K274210 were constitutively expressed.<br><br>
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Since the first experiment showed no or small amounts of product in cells grown to exponential phase the measurements were performed on cells incubated for 20 hours at 37degrees Celcius and 180rpm. The graphs obtained from the experiment are presented below:<br> <br>
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Since the first experiment showed little or no product in cells grown to exponential phase, the measurements were performed on cells incubated for 20 hours at 37degrees Celcius and 180rpm. The graphs obtained from the experiment are presented below:<br> <br>
[[Image: Team-SDU-denmark-Samples form top10 mg1655 and control.png |thumb|400px|center|UV-VIS spectre of six ''E. coli'' strains: Wild type TOP10, Wild type MG1655, TOP10-pSB1A2-K274210, MG1655-pSB1A2-K274210, TOP10-pSB1A2-K274210/pSB1C3-K343005 (double transformants), MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)]]
[[Image: Team-SDU-denmark-Samples form top10 mg1655 and control.png |thumb|400px|center|UV-VIS spectre of six ''E. coli'' strains: Wild type TOP10, Wild type MG1655, TOP10-pSB1A2-K274210, MG1655-pSB1A2-K274210, TOP10-pSB1A2-K274210/pSB1C3-K343005 (double transformants), MG1655-pSB1A2-K274210/pSB1C3-K343005 (double transformants)]]
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The graph also shows that cell material interferes with the UV-vis measurement where the retinal has the strongest absorption. Therefore, an organic separation is required prior to the measurements.<br>  
The graph also shows that cell material interferes with the UV-vis measurement where the retinal has the strongest absorption. Therefore, an organic separation is required prior to the measurements.<br>  
In order to assess the results, standard dilutions of beta-carotene and retinal were made and measured. The concentrations were 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standard dilutions were measured on the spectrophotometer. The resulting graphs are presented below:<br><br>
In order to assess the results, standard dilutions of beta-carotene and retinal were made and measured. The concentrations were 1mM, 100 µM, 50 µM, 25 µM, 10 µM, 5 µM, 1 µM, 100 nM and 10 nM. The standard dilutions were measured on the spectrophotometer. The resulting graphs are presented below:<br><br>
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[[Image: Team-SDU-denmark-Beta-carotene standarts.png|thumb|350px|left|UV-VIS of beta-carotene at different concentrations. The spectre looks reliable]][[Image: Team-SDU-denmark-Retinal standarts.png|thumb|315px|center|UV-VIS of retinal at different concentrations. The spectre does not look reliable]]
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[[Image: Team-SDU-denmark-Beta-carotene standarts.png|thumb|350px|center|UV-VIS of beta-carotene at different concentrations. The spectrum looks reliable]][[Image: Team-SDU-denmark-Retinal standarts.png|thumb|315px|center|UV-VIS of retinal at different concentrations. The spectrum does not look reliable]]
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The spectrum for the Beta-carotene looks normal and reliable, the standards for retinal however doesn’t look reliable. The spectrum for the three lowest concentrations appears to be give distorted and implies that something is interfering with the measurement.<br>  
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The spectrum for the beta-carotene looks normal and reliable, the standards for retinal however don’t look reliable. The spectrum for the three lowest concentrations appears to be give a distorted curve and implies that something is interfering with the measurement.<br>  
The measurement jumps rapidly from nearly nothing to the maximum absorbance, this might be because the concentration of retinal is to low to be measured as a spectrum, but the concentration of the individual derivates of retinal might be high enough to be measured.<br>
The measurement jumps rapidly from nearly nothing to the maximum absorbance, this might be because the concentration of retinal is to low to be measured as a spectrum, but the concentration of the individual derivates of retinal might be high enough to be measured.<br>
It might also just be that the concentrations are to low and something ells are influencing the measurements.<br>
It might also just be that the concentrations are to low and something ells are influencing the measurements.<br>
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Because of the error in the UV-vis spectre concerning retinal detection in both the standards and samples the next characterization experiments is preformed on a HPLC.<br>
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Because of the error in the UV-vis spectre concerning retinal detection in both the standards and samples the next characterization experiments were performed on a HPLC.<br>
   
   
All data can be seen under [http://2010.igem.org/wiki/images/7/7d/Team-SDU-Denmark-UV-Vis_determination_of_Carotenoids_and_polyene_chromophores_.zip Raw data]
All data can be seen under [http://2010.igem.org/wiki/images/7/7d/Team-SDU-Denmark-UV-Vis_determination_of_Carotenoids_and_polyene_chromophores_.zip Raw data]

Revision as of 00:40, 28 October 2010