|
|
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| Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments. | | Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments. |
| </figcaption> | | </figcaption> |
| + | <br> |
| + | Since the time of an iGEM project is limited to only one year, consequently only a limited amount of work can be |
| + | done in that time, which is even reduced by failing experiments and making mistakes in the lab. To overcome this |
| + | problem and increase the reproducibility and simultaneously raise the amount of experiments in the lab, we |
| + | automated plasmid purification on the OT-2. Using this protocol and making it open-source <b>(GitHub Link?)</b>, |
| + | we |
| + | achieved to parallelize work in the lab or make more time for public engagement, human practice, IHP or |
| + | everything else not directly lab-related, benefiting the whole iGEM community. This benefits will also be |
| + | translated beyond iGEM community such as in the amateur biohackers, enthusiasts, and students community and even |
| + | to research groups doing cutting-edge research.<br> |
| + | This idea started when we found out that there is also a great need in the industry for an automated cloning |
| + | workflow. Promega provided us with great advice <b>(Link to IHP)</b> and sponsored the Wizard® MagneSil® Plasmid |
| + | Purification System, QInstruments sponsored the BioShake D30-T elm and Opentrons sponsored their Magnetic |
| + | Module. Through our work aligned with the philosophy of iGEM for nurturing collaborations, we enabled |
| + | connections between these companies to achieve the true potential of their products. This kind of bridge would |
| + | not have been possible otherwise.<br> |
| + | <br> |
| + | Nevertheless, a massive amount of barriers had to be broken down. The shaker was a bit bigger than the space |
| + | normally occupied by modules in the OT-2 and needed stabilizing support, so it was obvious to design a |
| + | custom-made shaker adapter and print it with our own in-house 3D printer, which would keep the costs for the |
| + | automation of this workflow extremely low. Moreover, the 3D design will be publicly available in our GitHub |
| + | repository (LINK), which will make our solution accessible to everyone with access to a 3D printer.<br> |
| + | <br> |
| + | Additionally, we stumbled across serious problems with the calibration of our OT-2 and accessing the shaker with |
| + | the pipette. The BioShake D30-T elm is currently not a usual labware defined by Opentrons’, so we had to be |
| + | creative and come up with our own labware definition. Opentron is recently rolling out a major update from their |
| + | OT-2 3.9 to 4.0 firmware that includes a lot of paradigm changes, making it impossible for us to define it as a |
| + | decent custom labware. That is why we came up with the idea to use Opentrons’ internal coordinate system and |
| + | defining the required 96 Deep Well Plate on the shaker as coordinates. This facilitated accessing the shaker |
| + | with the pipette, being as precise as Opentrons’ own labware definitions, but a whole series of problems |
| + | followed, as we tried to use Opentrons’ pipette functions to transfer the chemicals. We managed these problems |
| + | as well, by defining our own Python functions, telling the pipette how to transfer liquids from and to the |
| + | defined shaker. In the end when running the script, one would not be able to tell the difference between the |
| + | labware and functions defined by us from the ones defined by Opentrons’.<br> |
| + | <br> |
| </figure> | | </figure> |
| </p> | | </p> |
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| and make a contribution to the cyano community by establishing essential/fixed standards in measurement. | | and make a contribution to the cyano community by establishing essential/fixed standards in measurement. |
| </p> | | </p> |
− | </article>
| + | <figure align=center> |
− | </section>
| + | <img style="height: 500px; width: 300px" |
− | <hr>
| + | src="https://static.igem.org/mediawiki/2019/b/bb/T--Marburg--opentrons_magnetic_module.JPG" |
− | <section class="section grid">
| + | alt="OT-2 left"> |
− | <div class="sub"
| + | <img style="height: 500px; width: 300px" |
− | onclick="popup('rbn1')">
| + | src="https://static.igem.org/mediawiki/2019/3/30/T--Marburg--opentrons_shaker.JPG" alt="OT-2 right"> |
− | <div class="sub-header"> | + | <figcaption style="max-width: 1400px"> |
− | <h1> | + | Fig.2 - Single-Channel pipette, magnetic module and shaker in action while performing the plasmid |
− | L I G H T<br>
| + | purification. |
− | M E A S U R E M E N T
| + | </figcaption> |
− | </h1>
| + | </figure> |
− | <hr>
| + | <br> |
− | </div>
| + | <p> |
− | <div class="sub-content">
| + | Putting the pieces together, we were able to translate the manual plasmid purification protocol provided by Nans |
− | <p>
| + | Bodet into an Opentrons protocol, being the very first of its kind. We pioneered a workflow for up to six |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | samples with the p300 Single-Channel Electronic Pipette and a scaled-up version for up to 48 samples with the |
− | </p>
| + | p300 8-Channel Electronic Pipette without having to intervene even once. This scalability provides important |
− | </div>
| + | flexibility for various kinds of experiments.<br> |
− | </div>
| + | <br> |
− | <div id="rbn1"
| + | In our process of developing and running the protocol we determined some problems on increasing the yield of our |
− | class="popup">
| + | plasmids. There was a large number of parameters that could be varied, changing the final concentration of the |
− | <div class="popup-container">
| + | plasmids. For example, we realized that the duration of lysis is paramount for the yield and success of plasmid |
− | <div class="popup-header">
| + | purification. Over-lysis will lead to a decrease in plasmid yield, whereas under-lysis will induce clumping of |
− | <h1 class="title">Light Measurement</h1>
| + | magnetic beads; thus failing the experiment. After a whole heap of plasmid purifications we managed to identify |
− | <button type="button"
| + | the most relevant parameters and improve the protocol in the best way possible.<br> |
− | onclick="hide('rbn1')">X</button>
| + | <br> |
− | </div>
| + | </p> |
− | <div class="popup-content"
| + | <figure align=center> |
− | style="text-align: justify; text-align-last: justify;">
| + | <img style="height: 700px; width: 600px" |
− | <section class="section">
| + | src="https://static.igem.org/mediawiki/2019/e/ea/T--Marburg--SingleChannelSetup.png" alt="OT-Layout left"> |
− | <p>Abstract?</p> | + | <img style="height: 700px; width: 600px" |
− | </section>
| + | src="https://static.igem.org/mediawiki/2019/d/df/T--Marburg--8channelSetup.png" alt="OT-Layout right"> |
− | <section class="section">
| + | <figcaption style="max-width: 1400px"> |
− | <div class="wrap-collabsible"
| + | Fig.3 - Final setup for the automated plasmid purification workflows in the OT-2. The left picture shows the |
− | style="margin-bottom: 25px;">
| + | setup for the single channel workflow, the right picture for the 8-channel workflow. |
− | <input id="collapsible1_1"
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− | class="lbl-toggle">
| + | poster="vorschaubild.jpg"></video> |
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| + | |
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| + | |
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| + | |
− | <h1> | + | |
− | F A C S
| + | |
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| + | |
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| + | |
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| + | |
− | 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> | + | |
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− | class="popup">
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| + | |
− | <button type="button"
| + | |
− | onclick="hide('rbn4')">X</button>
| + | |
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| + | |
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| + | |
− | <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>
| + | |
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| + | |
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| + | |
− | 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>
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− | <br>
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− | <b>Fluorescence-Activated Cell Sorting (FACS):</b><br>
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− | short abstract and link to the FACS-text of the measurement
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− | <br>
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− | <br>
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− | <b>Luminescence Measurement</b><br>
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− | <br>
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− | text
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− | </p>
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− | </div>
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− | </div>
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− | </div>
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− | <br>
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− | <div class="wrap-collabsible">
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− | <input id="collapsible4_2"
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− | class="toggle"
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− | type="checkbox">
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− | <label for="collapsible4_2"
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− | class="lbl-toggle">
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− | <h3 class="title"
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− | tyle="text-align: left; text-align-last: left;">Data analysis and evaluation
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− | </h3>
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− | </label>
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− | <div class="collapsible-content">
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− | <div class="content-inner"
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− | style="text-align: left; text-align-last: left;">
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− | <p>
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− | kein plan was man hier schreiben soll zum jetzigen standpunkt...
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− | For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for
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− | the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
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− | <br>
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− | Auswertung, Daten und Grafen darstellen?
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− | </p>
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− | </div>
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− | </div>
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− | </div>
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− | <div class="sub"
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− | onclick="popup('rbn5')">
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− | <div class="sub-header">
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− | <h1>
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− | G R O W T H<br>
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− | C U R V E S
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− | </h1>
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− | <hr>
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− | </div>
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− | <div class="sub-content">
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− | <p>
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− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
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− | class="popup">
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− | <div class="popup-container">
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− | <h1 class="title">Growth Curves</h1>
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− | <button type="button"
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− | onclick="hide('rbn5')">X</button>
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− | <div class="popup-content"
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− | style="text-align: justify; text-align-last: justify;">
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− | <p>
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− | Abstract?
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− | </p>
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− | <br>
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− | <br>
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− | <div class="wrap-collabsible">
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− | class="lbl-toggle">
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− | <h3 class="title">Unterprojekt1</h3>
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− | </label>
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− | <div class="collapsible-content">
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− | <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>
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− | <div class="wrap-collabsible">
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− | <input id="collapsible5_2"
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− | class="toggle"
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− | <h3 class="title">Unterprojekt2</h3>
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− | </label>
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− | <div class="collapsible-content">
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− | <div class="content-inner"
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− | style="text-align: left; text-align-last: left;">
| + | |
− | <p>
| + | |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | |
− | </p>
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− | </div>
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− | </div>
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− | </div>
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