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− | At this point it is important to mention that the light intensities in our incubator were not always set the same way: in the beginning we measured the light distribution with a planar light measurement device, using a conversion chart we acquired from Prof. Dr. Annegret Wilde from Freiburg to convert the values to theoretical spherical values, but after our insightful talk to Prof. Dr. James W. Golden (read here what else we learned from him[link to Golden Skype Call]) we hurried to get hold of a spherical measurement device to make sure we could accurately set the light intensities - and the difference was striking: the doubling time of our cultures increased by a huge amount which was an important step into the right direction for us.
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− | <h1>Fluorescence Activated Cell Sorting (FACS)<h1>
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| + | <div> |
| + | <div class="box-dark"> |
| + | <!--titel der seite--> |
| + | <h1 class="heading"> |
| + | M E A S U R E M E N T |
| + | </h1> |
| + | <hr class="line"> |
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| + | </div> |
| + | <div style="margin-top: 10vh;"> |
| + | <section class="section"> |
| + | <h1 class="title">Amplifying new standards in measurement</h1> |
| + | <!--Titel des textes--> |
| + | <p style="text-align: justify;"> |
| + | Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS) |
| + | </p> |
| + | </section> |
| + | <section class="section"> |
| + | <article> |
| + | <h1 class="title">Storytelling:</h1> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX |
| + | 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us |
| + | in front of many problems and questions. Especially the usage of different media, light conditions and other |
| + | cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. |
| + | Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. |
| + | Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic |
| + | microbiology and necessary to compare results with other scientists and reproduce their data. |
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to |
| + | show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore |
| + | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in |
| + | light measurement, evaluated different reporters???, established a measurement method and compared it to a |
| + | already known FACS measurement method (?). |
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. |
| + | So we had to measure the light conditions in our incubators and while doing this simple task the first |
| + | part of standardization began. We discovered that nearly every paper? is using different methods to measure |
| + | their light conditions and that it is a really complex and important procedure. So we got in contact with |
| + | cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following |
| + | popup we show different ways of measurement, their (dis-)advantages and different results depending on the |
| + | measuring instrument.<br> |
| + | Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. |
| + | In literature and while talking with different experts (IHP), we recognized that small deviations of these |
| + | parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as |
| + | a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters |
| + | that lead to the fastest growth speed.<br> |
| + | Another aspect was measuring the expression and characterize our part. Different possibilities were |
| + | discussed and after testing them we decided on two methods in our project (plate reader and FACs). One |
| + | approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate |
| + | readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br> |
| + | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In |
| + | contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its |
| + | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not |
| + | every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database |
| + | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the |
| + | difference in measurement methods.<br> |
| + | At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 |
| + | and make a contribution to the cyano community by establishing essential/fixed standards in measurement. |
| + | </p> |
| + | </article> |
| + | </section> |
| + | <hr> |
| + | <section class="section grid"> |
| + | <div class="sub" onclick="popup('rbn1')"> |
| + | <div class="sub-header"> |
| + | <h1> |
| + | L I G H T<br> |
| + | M E A S U R E M E N T |
| + | </h1> |
| + | <hr> |
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| + | <h1 class="title">Light Measurement</h1> |
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| + | Abstract? |
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| + | R E P O R T E R S |
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| + | F A C S |
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| + | <div class="sub-content"> |
| + | <p> |
| Fluorescence Activated Cell Sorting (FACS) is a flow cytometry measurement technique that separates single cells with different fluorescence characteristics. In this method you get accurate measuring results, because every single cell is analyzed on its own. FACS analysis can be used for cell sorting, fluorescence analysis of single cells and cell counting of different sample mixtures.<br> | | Fluorescence Activated Cell Sorting (FACS) is a flow cytometry measurement technique that separates single cells with different fluorescence characteristics. In this method you get accurate measuring results, because every single cell is analyzed on its own. FACS analysis can be used for cell sorting, fluorescence analysis of single cells and cell counting of different sample mixtures.<br> |
| In flow cytometry, the sample with the cells get hydrodynamically focused in a single stream. The cells arrange in a row, so that they can pass a laser one by one. The cells get excited by the laser and emit light at various wavelengths, which is then detected by a fluorescence analysator. The detector can detect and separate different fluorescence intensities at a definite range of wavelengths. Through this cells can be categorized by different fluorescent characteristics and if wanted separated into defined categories by a deflection system using electromagnetic fields. <br> | | In flow cytometry, the sample with the cells get hydrodynamically focused in a single stream. The cells arrange in a row, so that they can pass a laser one by one. The cells get excited by the laser and emit light at various wavelengths, which is then detected by a fluorescence analysator. The detector can detect and separate different fluorescence intensities at a definite range of wavelengths. Through this cells can be categorized by different fluorescent characteristics and if wanted separated into defined categories by a deflection system using electromagnetic fields. <br> |
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− | <h1> Light measurement <h1> | + | </p> |
− | | + | </div> |
− | At a very early stage of our project we noticed that standardization in the phototrophic community needs to have an overhaul to allow for reproducible experiments. As we started doing growth curves we used to determine the light intensity via a planar quantum sensor that can only absorb photons from an angle of approximately 120° and only counts photons having a wavelength between 400nm-700nm. Because of the way we setup our incubator the illumination was coming from two different light sources, which needed to be measured individually. While our first attempts included measuring the intensity by facing the quantum sensor at the lights respectively and then converting these values by a factor accounting for spherical flux of light. We then came up with the idea to search for a scalar radiometer that has a detection surface of nearly 4π steradian, can only measures photosynthetic active radiation. With the help of this method we used to determine the exact amount of µmol photonsm2s that can be used for photosynthesis. (400nm-700nm).
| + | </div> |
− | After we determined the light intensity via this method the doubling time of our strain drastically reduced. Doubling times from two hours we had before were now beaten and we achieved new lows of about 90 mins for the first time.
| + | <div id="rbn3" class="popup"> |
− | We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology. What we often time stumbled upon when we were looking for literature on our iGEM project was that the information on light intensity in these papers were often inconsistent. Oftentimes the only values on the intensity were given in the unit µEinstein, but the needed details on how that number was measured, was missing. So some people would measure the intensities with a planar device, others would determine them via a spherical quantum sensor.
| + | <div class="popup-container"> |
− | | + | <div class="popup-header"> |
− | During our skype call with James Golden he emphasized that a lot of experiments are simply not reproducible, because there is no way to tell how much light one has to expose their organisms to. Additionally we got the feedback of Dr. Nicolas Schmelling that even professional cultivation devices from companies which are specialized on building them, can not deliver consistent and even illumination.
| + | <h1 class="title">Fluorescence-Activated Cell Sorting (FACS)</h1> |
− | To go even further, we think that the spectrum of the respective lamp should also be considered when talking about standardization. The light spectrum of our two lamps look as follows.
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− | [evtl. nur Bild der Spektren]
| + | </div> |
− | Even though the standardization of the light quality seems to be a very hard task it should still be included in scientific works in order to give as much information as possible about the experimental setup.
| + | <div class="popup-content" style="text-align: justify; text-align-last: justify;"> |
− | We measured an equidistant grid of points at which we measured the average amount of photons (10 seconds) to minimize fluctuation. These data points were then interpolated with the help of a b spline surface to predict the amount of µmol photons at any given point of the incubator. This method is described in more detail on our model page.[Link zur Model page] We believe that the standardization of measuring light intensity has a huge impact in the field of phototrophic biology and immensely helps to create reproducible experimental setups.
| + | <p> |
− | We could show that light intensity had a big effect on reporter gene expression (FACS link)
| + | Abstract? |
− | This displays the importance of standardization especially if one want to characterize parts such as promoters RBS terminator or engineer even more complex designs like genetic circuits or synthetic metabolic pathways.
| + | </p> |
− | We propose a standardization of the light measurement process and inclusion of information, such as the way of measuring, light source and proper light intensities in every publication for phototrophic organisms.
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| + | P A R T<br> |
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| + | M E A S U R E M E N T |
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| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
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| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. |
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| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
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| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg |
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| + | 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 |
− | hr {
| + | different cultivation vessels and parameters - and revised them after evaluating the results. In the end |
− | display: block;
| + | we were able to establish a workflow specially designed for our methods to cultivate and characterize |
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| + | the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX 2973. |
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| + | Experimental Procedure |
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| + | The results of our part characterization were obtained by fluorescence and luminescence |
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| + | 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 |
− | .line {
| + | best growing conditions. It was logistically the best way to cultivate and measure the parts in |
− | margin: 1.5rem 0 !important; | + | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space |
− | margin-left: -40px !important;
| + | 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 |
− | </style>
| + | these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating |
− | <div>
| + | flasks and well-plates in the same incubator. After revising the workflow over and over we came to |
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| + | the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates |
− | <h1 class="heading">
| + | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. |
− | M E A S U R E M E N T
| + | Further it was necessary to use transparent wells to ensure every well with similar ight |
− | </h1>
| + | conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) |
− | <hr class="line">
| + | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an |
− | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg"
| + | important role in cultivation of well plates cause the realtive small volumes and high surfaces |
− | class="logo"
| + | (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is |
− | alt="Syntex Logo">
| + | essential to know the volume in the wells for measuring in the plate reader. Therefore we compared |
− | </div>
| + | different seals for the well plates and in the end we came to the conclusion that using a |
− | <div>
| + | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were |
− | <section class="section">
| + | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to |
− | </section>
| + | cultivate the cells for 2-3 days without losing significant amounts of medium. |
− | <section class="section" style="padding-top: 1;">
| + | <br> |
− | <div class="container">
| + | <br> |
− | <h1>Growth Curves</h1>
| + | <center>xxxx |
− | <div class="columns is-desktop">
| + | Fig x.:Schema vom Workflow</center> |
− | <div class="column">
| + | <br> |
− | <p style="margin-bottom: 1rem;">
| + | As described before we used the following workflow as shown in fig. XX to cultivate and measure |
− | <br> | + | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the |
− | <i>“Strength and growth come only through continuous effort and struggle.”</i> - Napoleon Hill | + | end of the triparental conjugation (LINK). For every part we picked 3 different colonies and |
− | <br> | + | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
− | <br> | + | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to |
− | Although this quote was certainly never meant in this way, it is quite fitting to our project, as the growth of our <i>Synechococcus elongatus</i> strain UTEX 2973 was one of the key aspects throughout the year.
| + | 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 |
− | Our goal to create the fastest phototrophic chassis was fueled by our unwavered dream of accelerated research on the multitude of mechanisms and possibilities that phototrophic organisms have to offer. We were quick to learn that this goal was not as close as we might have thought. On our way we encountered countless obstacles, some easier to overcome than others - one of the most resilient ones being the growth conditions we had to provide.
| + | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was |
− | Actually reaching the technical values we wanted was not the main issue, no, the hardest part was finding the holy grail of growth conditions, the perfect combination of parameters to cultivate our strain in.
| + | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while |
− | <br>
| + | evaluating the results (that will be used as a blank while ...). When all the cultures in the |
− | Digging through literature we found various different setups that were seemingly the “optimal growth conditions” for <i>S. elongatus</i> UTEX 2973 and it was apparent that in order to find the optimal conditions, we ultimately had to try all of them out by ourselves. So we set one of our biggest projects in motion, recording numerous different growth curves with many different parameters.
| + | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same |
− | Before calibrating key parameters like CO2 concentration, light intensity and temperature, we conducted some smaller trials on various other criteria, such as lid type, flask size, flask type and culture volume, as those are not heavily affected by the other parameters.
| + | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating |
− | Through these experiments, we could clearly identify a set that enabled the best growth for our chassis: plastic lids on 250ml erlenmeyer flasks with three chicanes and 50ml culture volume. Having fixed these initial parameters we set sail to the sea of endlessly variable growth conditions in hope to discover the true needs of <i>S. elongatus</i> UTEX 2973.
| + | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When |
− | As phototrophic chassis primarily require light and CO2 for their growth, those were the two parameters we were most interested in, but due to the UTEX 2973 strain being reportedly tolerant to higher temperatures than most other S.elongatus strains (Tan et al., 2018), this was another aspect to be tested. As time was scarce, we parallelized our measurements, meaning that while different temperatures or CO2 concentrations were put on trial we were able to compare the growth under different light intensities. | + | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
− | <br> | + | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three |
− | <br> | + | times. Following this workflow we were able to measure three biological parallels and |
− | Digging through literature we found various different setups that were seemingly the “optimal growth conditions” for <i>S. elongatus</i> UTEX 2973 and it was apparent that in order to find the optimal conditions, we ultimately had to try all of them out by ourselves. So we set one of our biggest projects in motion, recording numerous different growth curves with many different parameters.<br>
| + | two technical parallels for every biological parallel. It enabled us to have a good statistical |
− | Before calibrating key parameters like CO2 concentration, light intensity and temperature, we conducted some smaller trials on various other criteria, such as lid type, flask size, flask type and culture volume, as those are not heavily affected by the other parameters. <br>
| + | database and gives our results a stronger meaning/significance. While working with this workflow |
− | Through these experiments, we could clearly identify a set that enabled the best growth for our chassis: plastic lids on 250ml erlenmeyer flasks with three chicanes and 50ml culture volume. Having fixed these initial parameters we set sail to the sea of endlessly variable growth conditions in hope to discover the true needs of S.elongatus UTEX 2973.
| + | it was essential to keep the cultures in their exponential phase because it would significantly |
− | As phototrophic chassis primarily require light and CO2 for their growth, those were the two parameters we were most interested in, but due to the UTEX 2973 strain being reportedly tolerant to higher temperatures than most other S.elongatus strains (Tan et al., 2018), this was another aspect to be tested. As time was scarce, we parallelized our measurements, meaning that while different temperatures or CO2 concentrations were put on trial we were able to compare the growth under different light intensities.
| + | 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> | | </p> |
− | </div> | + | <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> |
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| + | <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> |
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