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| </figcaption> | | </figcaption> |
| </figure> | | </figure> |
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| </p> | | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <div class="sub" onclick="popup('rbn4')"> |
| + | <div class="sub-header"> |
| + | <h1> |
| + | P A R T<br> |
| + | M E A S U R E M E N T |
| + | </h1> |
| + | <hr> |
| + | </div> |
| + | <div class="sub-content"> |
| + | <p> |
| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | <div id="rbn4" class="popup"> |
| + | <div class="popup-container"> |
| + | <div class="popup-header"> |
| + | <h1 class="title">Part Measurement</h1> |
| + | <button type="button" onclick="hide('rbn4')">X</button> |
| + | </div> |
| + | <div class="popup-content"> |
| + | <p> |
| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg |
| + | Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement |
| + | method that suites such a large collection. Therefore we elaborated different workflows - containing |
| + | different cultivation vessels and parameters - and revised them after evaluating the results. In the end |
| + | we were able to establish a workflow specially designed for our methods to cultivate and characterize |
| + | the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX 2973. |
| + | </p> |
| + | <div class="wrap-collabsible"> |
| + | <input id="collapsible4_1" class="toggle" type="checkbox"> |
| + | <label for="collapsible4_1" class="lbl-toggle"> |
| + | <h3 class="title" style="text-align: left; text-align-last: left;"> |
| + | Experimental Procedure |
| + | </h3> |
| + | </label> |
| + | <div class="collapsible-content"> |
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| + | <p> |
| + | 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 |
| + | elaborate a cultivating and measuring workflow.<br> |
| + | For the cultivating workflow we tested different well plate formats and growing parameters for the |
| + | best growing conditions. It was logistically the best way to cultivate and measure the parts in |
| + | 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 |
| + | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small |
| + | 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 |
| + | these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating |
| + | flasks and well-plates in the same incubator. After revising the workflow over and over we came to |
| + | the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates |
| + | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. |
| + | Further it was necessary to use transparent wells to ensure every well with similar ight |
| + | conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) |
| + | 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 |
| + | (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is |
| + | 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 |
| + | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were |
| + | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to |
| + | cultivate the cells for 2-3 days without losing significant amounts of medium. |
| + | <br> |
| + | <br> |
| + | <center>xxxx |
| + | Fig x.:Schema vom Workflow</center> |
| + | <br> |
| + | 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 |
| + | end of the triparental conjugation (LINK). 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 |
| + | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to |
| + | OD<sub>730</sub>=0.6-0.8 they were inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells |
| + | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was |
| + | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while |
| + | evaluating the results (that will be used as a blank while ...). When all the cultures in the |
| + | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same |
| + | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating |
| + | 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 |
| + | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three |
| + | 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 |
| + | database and gives our results a stronger meaning/significance. While working with this workflow |
| + | it was essential to keep the cultures in their exponential phase because it would significantly |
| + | speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es |
| + | erst gar keine lag phase gibt).<br> |
| + | Concerning the measurement part we decided to transfer the cultures into black/white luminescence |
| + | 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 |
| + | eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for |
| + | measurement)<br> |
| + | <br> |
| + | <b>Fluorescence measurement:</b><br> |
| + | After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. |
| + | More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP |
| + | fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the |
| + | considered part. For measurement we created a program that measured the OD<sub>730</sub> and the |
| + | fluorescence of the wells.<br> |
| + | <br> |
| + | <center>fig XX (screenshot des messprogams)</center> |
| + | <br> |
| + | 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 |
| + | border of the well showed consistent results with small standard deviations (fig. XX). We used the |
| + | 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 |
| + | wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database |
| + | verlinken/als quelle?)<br> |
| + | <br> |
| + | <b>Fluorescence-Activated Cell Sorting (FACS):</b><br> |
| + | short abstract and link to the FACS-text of the measurement |
| + | <br> |
| + | <br> |
| + | <b>Luminescence Measurement</b><br> |
| + | <br> |
| + | text |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <br> |
| + | <div class="wrap-collabsible"> |
| + | <input id="collapsible4_2" class="toggle" type="checkbox"> |
| + | <label for="collapsible4_2" class="lbl-toggle"> |
| + | <h3 class="title" tyle="text-align: left; text-align-last: left;">Data analysis and evaluation |
| + | </h3> |
| + | </label> |
| + | <div class="collapsible-content"> |
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| + | <p> |
| + | kein plan was man hier schreiben soll zum jetzigen standpunkt... |
| + | For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for |
| + | the fluorescence measurement we used UTEX 2973 without a fluorescent protein. |
| + | <br> |
| + | Auswertung, Daten und Grafen darstellen? |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <div class="sub" onclick="popup('rbn5')"> |
| + | <div class="sub-header"> |
| + | <h1> |
| + | G R O W T H<br> |
| + | C U R V E S |
| + | </h1> |
| + | <hr> |
| + | </div> |
| + | <div class="sub-content"> |
| + | <p> |
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | <div id="rbn5" class="popup"> |
| + | <div class="popup-container"> |
| + | <div class="popup-header"> |
| + | <h1 class="title">Growth Curves</h1> |
| + | <button type="button" onclick="hide('rbn5')">X</button> |
| + | </div> |
| + | <div class="popup-content" style="text-align: justify; text-align-last: justify;"> |
| + | <p> |
| + | Abstract? |
| + | </p> |
| + | <br> |
| + | <br> |
| + | <div class="wrap-collabsible"> |
| + | <input id="collapsible5_1" class="toggle" type="checkbox"> |
| + | <label for="collapsible5_1" class="lbl-toggle"> |
| + | <h3 class="title">Unterprojekt1</h3> |
| + | </label> |
| + | <div class="collapsible-content"> |
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| + | <p> |
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <br> |
| + | <div class="wrap-collabsible"> |
| + | <input id="collapsible5_2" class="toggle" type="checkbox"> |
| + | <label for="collapsible5_2" class="lbl-toggle"> |
| + | <h3 class="title">Unterprojekt2</h3> |
| + | </label> |
| + | <div class="collapsible-content"> |
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| + | <p> |
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| + | </p> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </section> |
| + | </div> |
| + | </div> |
| + | </body> |
| + | |
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