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| <p> | | <p> |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. | + | Fluorescence Reporters |
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| <div class="popup-content" style="text-align: justify; text-align-last: justify;"> | | <div class="popup-content" style="text-align: justify; text-align-last: justify;"> |
| <p> | | <p> |
− | Abstract?
| + | When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable elements to characterize BioBricks. For a good characterization a suitable reporter is required. But reporters can be more than just merely a detection tool for transcriptional activity but they can also give a deeper insight into cellular conditions beyond the genetic context. We provide a diverse set of reporters not only for the purpose of describing genetic tools but also for the sensing of a variety of parameters which are crucial for cyanobacteria. |
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− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
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− | <div class="content-inner" style="text-align: left; text-align-last: left;">
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− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
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− | </p>
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| The results of our part characterization were obtained by fluorescence and luminescence | | The results of our part characterization were obtained by fluorescence and luminescence |
− | measurements (of what?). But before the party could be measured we had to | + | measurements. But before the party could be measured we had to |
| elaborate a cultivating and measuring workflow.<br> | | elaborate a cultivating and measuring workflow.<br> |
| For the cultivating workflow we tested different well plate formats and growing parameters for the | | For the cultivating workflow we tested different well plate formats and growing parameters for the |
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| well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space | | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space |
| in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus | | in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus |
− | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small | + | elongatus</i> UTEX 2973 under our conditions since the cultures showed small |
| clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of | | clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of |
| the incubator was limited whereas cultures in flasks had to be incubated at the same time and | | the incubator was limited whereas cultures in flasks had to be incubated at the same time and |
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| growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an | | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an |
| important role in cultivation of well plates cause the realtive small volumes and high surfaces | | important role in cultivation of well plates cause the realtive small volumes and high surfaces |
− | (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is | + | . Further it is |
| essential to know the volume in the wells for measuring in the plate reader. Therefore we compared | | essential to know the volume in the wells for measuring in the plate reader. Therefore we compared |
| different seals for the well plates and in the end we came to the conclusion that using a | | different seals for the well plates and in the end we came to the conclusion that using a |
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| As described before we used the following workflow as shown in fig. XX to cultivate and measure | | As described before we used the following workflow as shown in fig. XX to cultivate and measure |
| our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the | | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the |
− | end of the triparental conjugation (LINK). For every part we picked 3 different colonies and | + | end of the triparental conjugation. For every part we picked 3 different colonies and |
| inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates | | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
| we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to | | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to |
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| technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When | | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When |
| the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred | | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
− | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three | + | into a 96-well-plate. Every well of the 24-well-plate was measured three |
| times. Following this workflow we were able to measure three biological parallels and | | times. Following this workflow we were able to measure three biological parallels and |
| two technical parallels for every biological parallel. It enabled us to have a good statistical | | two technical parallels for every biological parallel. It enabled us to have a good statistical |
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| is measured in white ones. We measured in 96-well-plates because it enabled us to measure every | | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every |
| part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure | | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure |
− | eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for | + | eight parts in only one plate. (four 24-well-plates lead into one 96-well-plate for |
| measurement)<br> | | measurement)<br> |
| <br> | | <br> |
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| fluorescence of the wells.<br> | | fluorescence of the wells.<br> |
| <br> | | <br> |
− | | + | OD measure with plate reader: Settings: 730 nm, 3 measuring points (circle) <br> Fluorescence Settings: Excitation 488 nm, Emission 518 nm size 2x2 (circle), frame 1200 μm strengthener:optimal |
| <br> | | <br> |
| In order to measure the OD in each well we determined the absorption at 730 nm. Further we | | In order to measure the OD in each well we determined the absorption at 730 nm. Further we |
| measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the | | measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the |
− | border of the well showed consistent results with small standard deviations (fig. XX). We used the | + | border of the well showed consistent results with small standard deviations. We used the |
| same settings of the multiple measurement for the fluorescence measurement. While using sYFP as | | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as |
| signal for our part measurement we have set the emission wavelength to 515 nm and the excitation | | signal for our part measurement we have set the emission wavelength to 515 nm and the excitation |
− | wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database | + | wavelength to 527 nm, fitting the exact wavelengths of the sYFP.<br> |
− | verlinken/als quelle?)<br>
| + | |
| <br> | | <br> |
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