Latest revision as of 02:41, 14 December 2019

G I A N T
J A M B O R E E

Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. We loved watching the other team's presentations and listening to their detailed explanations at their posters. Thereby, we realised once again how broad the field of Synthetic Biology is and how big of an impact this field already is and how it will be even bigger in the future. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year.

Our presentation was one of the first on the first day. The room was so crowded, people were even standing in the doorway to watch our presentation! Here you have the possibility to see the presentation of our team at the Giant Jamboree.

Afterwards, most of the time we had to stay with three or more people near our poster, because so many people were interested in our project. Additionally, Keoni Gandall and Kristin Ellis paid us a visit at our Exhibition Space booth. They were really impressed by our custom-made hardware.

Our team at the roof of the Opentrons building, during our visit in New York City

Kristin was so happy about our work on the OT-2 Colony Picker Unit that she invited us to their company headquarters in New York City! When we went to Opentrons in New York City Kristin showed us their offices and labs, explained us how they work at Opentrons and we presented our Colony Picker Unit to several staff members, sharing our thoughts and ideas.

Working independently and in collaborations on our own project, joining the Giant Jamboree together as a team and getting in touch with all these different, amazing people meant a lot to us and we are really thankful to have made these experiences! Finally we want to thank everybody who helped us gain a silver medal in this awesome competition! We are happy for this amazing time and to be a part of this great community!

Metabolic MEP Pathway — Fig. 1- Overview of the MEP-Pathway.
Enzymes are marked blue and Green.
Limonene and farnesene Synthase are summarised as TPS.
Modified after Lin et al 2016

Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene synthase. Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (Lin et al., 2016).

Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year.

Storytelling:

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.

asas — Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus UTEX 2973 Absorption spectra.

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 (?).

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.
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.
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.
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.
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.

L I G H T
M E A S U R E M E N T

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

R E P O R T E R S

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

F A C S

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

P A R T
M E A S U R E M E N T

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.

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 Synechococcus elongatus UTEX 2973.

Experimental Procedure

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.
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 Synechococcus elongatus 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.

xxxx Fig x.:Schema vom Workflow
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 OD₇₃₀=0.6-0.8 they were inoculated to 1.0 mL of OD₇₃₀=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 OD₇₃₀= 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 OD₇₃₀=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 OD₇₃₀=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).
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)

Fluorescence measurement:
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 OD₇₃₀ and the fluorescence of the wells.

fig XX (screenshot des messprogams)
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?)

Fluorescence-Activated Cell Sorting (FACS):
short abstract and link to the FACS-text of the measurement

Luminescence Measurement

text

Data analysis and evaluation

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.
Auswertung, Daten und Grafen darstellen?

G R O W T H
C U R V E S

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

Abstract?

Unterprojekt1

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

Unterprojekt2

Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.

One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds with CO₂ and light as a resource. As a target, we thought about molecules, which also tackle one of the most important topic: the climate change.

Planes are considered to be one of the most environmentally damaging transport devices commonly used (Borken-Kleefeld et al., 2010). A quick estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO₂ was released from the flights. To encounter this problem, we chose to set our focus on farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50% limonene, 40% farnesene) from Amyris (Brennan et al., 2015). This fuel has proven to be a suitable alternative to chemically produced, oil based jet fuels

Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3). The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene synthase. Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in cyanobacteria (Lin et al., 2016).

As a the first step we decided to heterologous express the limonene and the farnesene synthase to establish overexpression strains. We used the limonene synthase from Lavandula angustifolia and the Farnesen synthase from Actinidia deliciosa and codon optimised both enzymes. To redirect the flux into the MEP-Pathway we decided to overexpress the E.Coli proteins DXS, IDI and IspA. These targets were chosen on based previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942 without significant impairing the growth rate (Choi et al.,2016). Surprisingly, this suggest strong capacities of production in Cyanobacteria.

We chose the DXS, as many pathways are regulated on the first committing step. The IDI, would convert DMAPP to IPP, which is necessary to balance both pools, which is especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour of IPP is required.

IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a farnesene production strain. To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a phenylalanine (F). This would lead to a threefold increase in activity in vitro (Xiang et al., 2016). Also it would be advisable to remove the allosteric feedback regulation of the DXS (Banerjee et al.,2016).

Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the extracellular diffusion rate is enough to prevent intracellular and potential toxic accumulation. On the other hand, a nontoxic overlay to catch the molecules is required, for example dodecan can be used. (Lee et al., 2017). An alternative supply with CO2 is also required, therefore we copied the system from Lee et al. and introduced a small tube with holes into the Medium.

To balance the pathway we decided to use a weak promotor (BBa_J23103) for the ispA, as competition over FPP could lead to a heavy growth impact (Lin et al., 2016). As IDI is an Isomerase, only a comparably small amount of protein should be sufficient for a enhanced effect, so Promotor BBa_J23110 was chosen. Because the DXS is the a potential bottle neck in Terpene Production, we chose the Promotor BBa_J23111.

Sadly, we weren´t able to test our constructs in vivo and dropped this side project due to time reasons. As of now the submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the registry ( K3228051 and K3228052).

@@ Line 1: / Line 1: @@
 {{Marburg}}
-{{Marburg/team.css}}
 <html>
-   <style>
+   <div>
-     .columns:not(:last-child) {
+     <div class="box-dark">
-      margin-bottom: calc(1.5rem - .5rem) !important;
-    }
-     @media (max-width: 925px) {
+     <h1 class="heading" style="
-       .item-content {
+     line-height: 1;
-        max-width: unset !important;
+">
-        width: unset !important;
-       }
+        G I A N T<br> J A M B O R E E
-    }
+       </h1>
+      <hr class="line">
+      <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo">
+    </div>
+    <div style="margin-top: 10vh;">
+       <section class="section">
+      <div style="display: flex; flex-direction: row;">
-    .column {
+<p style="margin-bottom: 1em;">
-      padding-top: 0;
+         Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. We loved watching the other team's presentations and listening to their detailed explanations at their posters. Thereby, we realised once again how broad the field of Synthetic Biology is and how big of an impact this field already is and how it will be even bigger in the future. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year.
-      padding-bottom: 0;
-      font-size: medium;
-    }
-    hr {
+</p>
-      display: block;
-      height: 1px;
-      border: 0;
-      border-top: 2px solid #69a555;
-      padding: 0;
-      margin: 1em 0;
-    }
-    .is-3 {
+                        <figure style="text-align: center; margin-left: 25px; max-width: 42000px">
-      width: 17.4% !important;
+                          <img src="https://static.igem.org/mediawiki/2019/5/53/T--Marburg--GiantJamboree_2019_Grouppicture.jpg"
-    }
+            alt="Our team at the Giant Jamboree">
+          <figcaption style="text-align: center;">
+            Our team at the Giant Jamboree
+          </figcaption>
+                        </figure>
-    .item {
+                        </div>
-      height: auto;
-      width: 100%;
-      margin-bottom: 1em;
-      display: flex;
-      flex-direction: row;
-    }
-    @media screen and (max-width: 925px) {
-      .item {
-        flex-direction: column !important;
-      }
-      .item-content {
-        padding-left: 0 !important;
-      }
-      .is-3 {
-        width: unset !important;
-      }
-      .item-column {
+<p style="margin-bottom: 1em;">
-        padding-left: .75rem !important;
+<br> <br> Our presentation was one of the first on the first day. The room was so crowded, people were even standing in the doorway to watch our presentation! Here you have the possibility to see the presentation of our team at the Giant Jamboree.
-      }
+</p>
-     }
+<div style="
+    position: relative;
+    height: 0;
+    width: 100%;
+    padding-bottom: 56.25%;
+">
+     <iframe src="https://www.youtube.com/embed/CwasIzE0R8o" frameborder="0" allow="accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture" allowfullscreen="" style="position: absolute;width: 100%;height: 100%;top: 0;left: 0;"></iframe>
+</div>
-    .item-content {
+<br>
-      max-width: calc(100% - 200px);
-      padding-left: 1em;
-      padding-right: 1em;
-      width: calc(100% - 200px);
-    }
-    .item-column {
-      padding-left: 0;
-    }
-    .container {
+<br>
-      display: flex;
+<p style="margin-bottom: 1em;">
-      justify-content: center;
+<br> Afterwards, most of the time we had to stay with three or more people near our
-    }
+ <a style="padding: 0" href="https://static.igem.org/mediawiki/2019/2/2a/T--Marburg--Poster_2019_Marburg.pdf" target="_blank">poster</a>, because so many people were interested in our project.
+Additionally, Keoni Gandall and Kristin Ellis paid us a visit at our Exhibition Space booth. They were really impressed by our custom-made hardware.
+ </p>
+<figure style="float:right; margin-left: 25px; text-align: center;">
+          <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/9/93/T--Marburg--OpentronsVisitinNY_Grouppicture.jpg"
+            alt="Our team at the roof of the Opentrons building, during our visit in New York City">
+          <figcaption style="text-align: center;">
+            Our team at the roof of the Opentrons building, during our visit in New York City
+          </figcaption>
+                        </figure>
+ <p style="margin-bottom: 1em;">
+Kristin was so happy about our work on the OT-2 Colony Picker Unit that she invited us to their company headquarters in New York City! When we went to Opentrons in New York City Kristin showed us their offices and labs, explained us how they work at Opentrons and we presented our Colony Picker Unit to several staff members, sharing our thoughts and ideas.
+ </p>
-    b {
-      font-size: large;
-    }
-    p {
-      margin-bottom: 1rem;
-      max-width: 750px;
-    }
-    .picture_group {
+ <p style="margin-bottom: 1em;">
-      margin: auto;
+Working independently and in collaborations on our own project, joining the Giant Jamboree together as a team and getting in touch with all these different, amazing people meant a lot to us and we are really thankful to have made these experiences! Finally we want to thank everybody who helped us gain a silver medal in this awesome competition! We are happy for this amazing time and to be a part of this great community!
-      border-radius: 8px;
+        </p>
-      display: block;
-      width: 800px;
+<br><br><br>
-    }
-    .text_group {
-      font-size: large !important;
-      padding-left: 2em;
-      padding-right: 2em;
-      padding-top: 1em;
-      text-align: center !important;
-    }
-    .picture {
-      height: 200px;
-      width: 200px;
-      border-radius: 8px;
-      transition: background .25s ease-in-out;
-    }
-    .name {
-      font-size: x-large !important;
-    }
-    .picture_vinca {
+        <figure style="float:right; margin-left: 25px; text-align: center;">
-      background: url("https://static.igem.org/mediawiki/2019/2/2a/T--Marburg--Team_Vinca1.jpg");
+          <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/7/7d/T--Marburg--Metabolic--MEP-pathway.png"
-    }
+            alt="Metabolic MEP Pathway">
+          <figcaption style="text-align: center;">
+            Fig. 1- Overview of the MEP-Pathway.<br>
+            Enzymes are marked blue and Green.<br>
+            Limonene and farnesene Synthase are summarised as TPS.<br>
+            Modified after Lin et al 2016
+          </figcaption>
+        </figure>
+        <p  style="margin-bottom: 1em;">
+          Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure 3).
+          The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle (directly
+          as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl pyrophosphate (IPP) and it´s
+          isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and Farnesyl pyrophosphate (FPP). GPP serves
+          as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas FPP is a substrate for the plant derived Farnesene
+          synthase.
+          Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway in
+          cyanobacteria (<a style="padding: 0" href="https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>,
+</a>).
+        </p>
-    .picture_vinca:hover {
-      background: url("https://static.igem.org/mediawiki/2019/8/8d/T--Marburg--Team_Vinca2.jpg");
-    }
-    .picture_cedric {
-      background: url("https://static.igem.org/mediawiki/2019/9/9d/T--Marburg--Team_Cedric1.jpeg");
-    }
-    .picture_cedric:hover {
-      background: url("https://static.igem.org/mediawiki/2019/f/f8/T--Marburg--Team_Cedric2.jpg")
-    }
-    .picture_rene {
-      background: url("https://static.igem.org/mediawiki/2019/6/68/T--Marburg--Team_Rene1.jpg");
-    }
-    .picture_rene:hover {
-      background: url("https://static.igem.org/mediawiki/2019/2/23/T--Marburg--Rene2.jpg");
-    }
-    /* TODO torben photo */
-    .picture_torben {
-      background: url("https://proxy.duckduckgo.com/iu/?u=http%3A%2F%2Fwww.theplantationshutterco.com%2Fimages%2Fmade%2Fmedia%2Fimages%2Fprofile_placeholder_200_200_s_c1.png&f=1&nofb=1");
-    }
-    .picture_torben:hover {
-      background: url("https://proxy.duckduckgo.com/iu/?u=https%3A%2F%2Fi.stack.imgur.com%2FWmvM0.png&f=1&nofb=1");
-    }
-    .picture_pascal {
-      background: url("https://static.igem.org/mediawiki/2019/9/93/T--Marburg--Team_Pascal1.jpg");
-    }
-    .picture_pascal:hover {
-      background: url("https://static.igem.org/mediawiki/2019/7/71/T--Marburg--Team_Pascal2.jpg");
-    }
-    .picture_hinrik {
-      background: url("https://static.igem.org/mediawiki/2019/8/88/T--Marburg--Team_Hinrik1.jpg");
-    }
-    .picture_hinrik:hover {
-      background: url("https://static.igem.org/mediawiki/2019/4/48/T--Marburg--Team_Hinrik2.jpg");
-    }
-    .picture_michael {
-      background: url("https://static.igem.org/mediawiki/2019/c/c2/T--Marburg--Team_Michael1.jpg");
-    }
-    .picture_michael:hover {
+<figure style="float:right; margin-left: 25px; text-align: center;">
-      background: url("https://static.igem.org/mediawiki/2019/a/a0/T--Marburg--Team_Michael2.jpg");
+          <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/9/93/T--Marburg--OpentronsVisitinNY_Grouppicture.jpg"
-    }
+            alt="Our team at the roof of the Opentrons building, during our visit in New York City">
+          <figcaption style="text-align: center;">
+            Our team at the roof of the Opentrons building, during our visit in New York City
+          </figcaption>
+        </figure>
+        <p  style="margin-bottom: 1em;">
-    .picture_jonas {
+        <p style="margin-bottom: 1em;">
-      background: url("https://static.igem.org/mediawiki/2019/a/a8/T--Marburg--Team_Jonas1.jpg");
+         Traditionally, we presented our project at the Giant Jamboree in Boston. It was a lot of fun to meet all the other iGEM teams and to talk and discuss with like-minded people. With our presentation, poster and a booth at the Exhibition Space we showed what we were able to achieve in the last year.
-    }
-    .picture_jonas:hover {
+</p>
-      background: url("https://static.igem.org/mediawiki/2019/6/60/T--Marburg--Team_Jonas2.jpg");
-    }
-    .picture_maurice {
-      background: url("https://static.igem.org/mediawiki/2019/7/7c/T--Marburg--Team_Maurice1.jpg");
-    }
-    .picture_maurice:hover {
-      background: url("https://static.igem.org/mediawiki/2019/8/8d/T--Marburg--Team_Maurice2.jpg");
-    }
-    .picture_marian {
-      background: url("https://static.igem.org/mediawiki/2019/c/ce/T--Marburg--Team_Marian1.jpg");
-    }
-    .picture_marian:hover {
+       </section>
-       background: url("https://static.igem.org/mediawiki/2019/5/5e/T--Marburg--Team_Marian2.jpg");
+       <section class="section">
-    }
+        <article>
+          <h1 class="title">Storytelling:</h1>
-    .picture_paula {
+          <p style="text-align: justify; margin-bottom: 1em;">
-       background: url("https://static.igem.org/mediawiki/2019/8/80/T--Marburg--Team_Paula1.jpg");
+            We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX
-    }
+, 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
-    .picture_paula:hover {
+            cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers.
-      background: url("https://static.igem.org/mediawiki/2019/6/6e/T--Marburg--Team_Paula2.jpg");
+            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.
-    .picture_lars {
+          </p>
-      background: url("https://static.igem.org/mediawiki/2019/c/ca/T--Marburg--Team_Lars1.jpg");
+          <figure style="float:right; margin-left: 25px;">
-    }
+            <img style="height: 719px; width: 1001px;" src="https://2019.igem.org/File:T--Marburg--Metabolic--MEP-pathway.png" alt="asas">
+            <figcaption style="max-width: 550px; text-align: center">
-    .picture_lars:hover {
+              Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus
-      background: url("https://static.igem.org/mediawiki/2019/4/42/T--Marburg--Team_Lars2.jpg");
+              UTEX
-    }
+Absorption spectra.
+            </figcaption>
-    .picture_wihan {
+          </figure>
-      background: url("https://static.igem.org/mediawiki/2019/b/bc/T--Marburg--Team_Wihan1.jpg");
+          <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
-    .picture_wihan:hover {
+            we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
-      background: url("https://static.igem.org/mediawiki/2019/5/59/T--Marburg--Team_Wihan2.jpg");
+            light measurement, evaluated different reporters???, established a measurement method and compared it to a
-    }
+            already known FACS measurement method (?).
+          </p>
-    .picture_annsophie {
+          <p style="text-align: justify; margin-bottom: 1em;">
-      background: url("https://static.igem.org/mediawiki/2019/1/1e/T--Marburg--Team_Annsophie1.jpg");
+            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
-    .picture_annsophie:hover {
+             their light conditions and that it is a really complex and important procedure. So we got in contact with
-      background: url("https://static.igem.org/mediawiki/2019/9/99/T--Marburg--Team_Annsophie2.jpg");
+            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>
-    .picture_ting {
+             Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed.
-      background: url("https://static.igem.org/mediawiki/2019/c/ce/T--Marburg--Team_Ting1.jpg");
+             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
-    .picture_ting:hover {
+            that lead to the fastest growth speed.<br>
-      background: url("https://static.igem.org/mediawiki/2019/1/11/T--Marburg--Team_Ting2.jpg");
+            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
-    .picture_robin {
+             readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br>
-      background: url("https://static.igem.org/mediawiki/2019/5/57/T--Marburg--Team_Robin1.jpg");
+             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
-    .picture_robin:hover {
+             every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database
-      background: url("https://static.igem.org/mediawiki/2019/0/06/T--Marburg--Team_Robin2.jpg");
+             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
-    .picture_chun {
+            and make a contribution to the cyano community by establishing essential/fixed standards in measurement.
-      background: url("https://static.igem.org/mediawiki/2019/4/48/T--Marburg--Team_Chun1.jpg");
+          </p>
-    }
+        </article>
+      </section>
-    .picture_chun:hover {
+      <hr>
-      background: url("https://static.igem.org/mediawiki/2019/e/ee/T--Marburg--Team_Chun2.jpg");
+      <section class="section grid">
-    }
+        <div class="sub" onclick="popup('rbn1')">
+          <div class="sub-header">
-    .picture_johanna {
+             <h1>
-      background: url("https://static.igem.org/mediawiki/2019/b/b0/T--Marburg--Team_Johanna1.jpg");
+              L I G H T<br>
-    }
+              M E A S U R E M E N T
+             </h1>
-    .picture_johanna:hover {
+             <hr>
-      background: url("https://static.igem.org/mediawiki/2019/7/75/T--Marburg--Team_Johanna2.jpg");
-    }
-    .picture_joana {
-      background: url("https://static.igem.org/mediawiki/2019/5/5c/T--Marburg--Team_Joana1.jpg");
-    }
-    .picture_joana:hover {
-      background: url("https://static.igem.org/mediawiki/2019/e/e6/T--Marburg--Team_Joana2.jpg");
-    }
-    .picture_jana {
-      background: url("https://static.igem.org/mediawiki/2019/9/9e/T--Marburg--Team_Jana1.jpg");
-    }
-    .picture_jana:hover {
-      background: url("https://static.igem.org/mediawiki/2019/d/d7/T--Marburg--Team_Jana2.jpg");
-    }
-    .picture_julia {
-      background: url("https://static.igem.org/mediawiki/2019/8/80/T--Marburg--Team_Julia1.jpg");
-    }
-    .picture_julia:hover {
-      background: url("https://static.igem.org/mediawiki/2019/c/cc/T--Marburg--Team_Julia2.jpg");
-    }
-    .picture_erik {
-      background: url("https://static.igem.org/mediawiki/2019/8/8a/T--Marburg--Team_Erik1.jpg");
-    }
-    .picture_erik:hover {
-      background: url("https://static.igem.org/mediawiki/2019/0/00/T--Marburg--Team_Erik2.jpg");
-    }
-  </style>
-  <div>
-    <section style="margin-bottom: 1em;">
-      <img class="picture_group"
-        src="https://static.igem.org/mediawiki/2019/thumb/9/9f/T--Marburg--Team_Group.jpg/800px-T--Marburg--Team_Group.jpg" />
-      <p class="text_group" style="max-width: 1000px; margin: auto;">
-        Meet the iGEM Team Marburg 2019! Our Team consists of 22 students including our
-        instructor and advisors. We came together from various fields such as biology, mathematics,
-        chemistry, physics, pharmaceutics and computer sciences, working interdisciplinary on our
-        project 'Syntex'. We want to thank our advisors, instructors and our two Principal
-        Investigators for all the support!
-      </p>
-    </section>
-    <section class="container">
-      <div style="display: flex; flex-direction: column;">
-        <hr>
-        <h1 class="title">Team Leaders</h1>
-        <div class="item">
-          <div class="picture picture_vinca"></div>
-          <div class="item-content">
-             <p class="name">
-              VINCA SEILER
-             </p>
-             <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-             </div>
-             <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Molecular and Cellular Biology
-              </div>
-             </div>
-             <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Strain engineering
-              </div>
-             </div>
-             <b>How I 'use' the sunlight in my leisure time:</b><br>
-             <p>
-              I love to picnic in the sun with food, music, friends and more food… cause light and CO<sub>2</sub> are
-              not enough for me.
-             </p>
-             <b>What I gained in this iGEM year:</b><br>
-             <p>
-              Soft Skills, Hard Skills, Medium Skills and Fluffy Skills.<br>
-              Besides this a lot of fun time in the lab, on Meet-UPs and with the team in general.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Time with my friends and family.<br>
-              Dear Grandparents, I’m still alive! And also maybe a healthy lifestyle.
-            </p>
-             <b>A fun fact about me:</b><br>
-             <p>
-              Sometimes I pray to the DNA-God while doing a PCR or Gibson-Assembly...
-             </p>
            </div>
-        </div>
+           <div class="sub-content">
-        <div class="item">
+             <div>
-           <div class="picture picture_cedric"></div>
+               Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-          <div class="item-content">
-            <p class="name">
-              CEDRIC BRINKMANN
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Mathematics
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                DryLab
-              </div>
              </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              When I'm not hiding from the sun and playing with the OT-2, I like to ride my bicycle or barbecue with
-              friends at the Lahn.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Caffeine addiction.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My train of thought...ummmm what?!
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I’m practically addicted to sunflower seeds.
-            </p>
            </div>
          </div>
-        <hr>
+         <div id="rbn1" class="popup">
-        <h1 class="title">Advisor - Instructor</h1>
+           <div class="popup-container">
-         <div class="item">
+             <div class="popup-header">
-          <div class="picture picture_rene"></div>
+               <h1 class="title">Light Measurement</h1>
-           <div class="item-content">
+               <button type="button" onclick="hide('rbn1')">X</button>
-            <p class="name">
-              RENÉ INCKEMANN
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-             <div class="columns">
+             <div class="popup-content" style="text-align: justify; text-align-last: justify;">
-               <div class="column is-3">
+               <section class="section">
-                <b>Field of Study:</b>
+                Abstract?
-              </div>
+              </section>
-              <div class="column item-column">
+              <section class="section">
-                 Master’s student Molecular and Cellular Biology
+                <div class="wrap-collabsible" style="margin-bottom: 25px;">
-               </div>
+                  <input id="collapsible1_1" class="toggle" type="checkbox">
+                  <label for="collapsible1_1" class="lbl-toggle">
+                    Unterprojekt1
+                    <hr style="width: unset;">
+                  </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 class="wrap-collabsible">
+                  <input id="collapsible1_2" class="toggle" type="checkbox">
+                  <label for="collapsible1_2" class="lbl-toggle">
+                    Unterprojekt2
+                    <hr style="width: unset;">
+                  </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>
+               </section>
              </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              When I am not doing stuff for iGEM or my master thesis, I like to make music, play guitar and when
-              the sun is shining I ride my motorbike.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Every new iGEM year is completely different, because of the different people.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Being focused on my Master thesis.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              Some people say, that I am addicted to iGEM.
-            </p>
            </div>
          </div>
-        <hr>
+         <div class="sub" onclick="popup('rbn2')">
-        <h1 class="title">Advisors</h1>
+           <div class="sub-header">
-         <div class="item">
+             <h1>
-          <div class="picture picture_torben"></div>
+               R E P O R T E R S
-           <div class="item-content">
+             </h1>
-             <p class="name">
+             <hr>
-               TORBEN GUTERMUTH
-             </p>
-             <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Chemistry
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I avoid it to not ruin my cellar tan.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              A distinct love for nihilistic black metal while working.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              The ability to work with anything that is not black or melodic death metal.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I can write without looking at the paper.
-            </p>
            </div>
-        </div>
+           <div class="sub-content">
-        <div class="item">
+             <div>
-           <div class="picture picture_pascal"></div>
+               Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-          <div class="item-content">
-            <p class="name">
-              PASCAL PFISTER
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">25</div>
              </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Synthetic Microbiology
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Grow plants.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              <!--TODO pascal gain-->
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              A slot for a bachelor student.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              There are more microbes in my beard than in our cultures.
-            </p>
            </div>
          </div>
-        <hr>
+         <div id="rbn2" class="popup">
-        <h1 class="title">Members</h1>
+           <div class="popup-container">
-         <div class="item">
+             <div class="popup-header">
-          <div class="picture picture_hinrik"></div>
+               <h1 class="title">Reporter</h1>
-           <div class="item-content">
+               <button type="button" onclick="hide('rbn2')">X</button>
-            <p class="name">
-              HINRIK PLAGGENBORG
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-             <div class="columns">
+             <div class="popup-content" style="text-align: justify; text-align-last: justify;">
-               <div class="column is-3">
+              <p>
-                 <b>Field of Study:</b>
+                Abstract?
+              </p>
+              <br>
+              <br>
+               <div class="wrap-collabsible">
+                 <input id="collapsible2_1" class="toggle" type="checkbox">
+                <label for="collapsible2_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>
-               <div class="column item-column">
+              <br>
-                 Master’s student Molecular and Cellular Biology
+               <div class="wrap-collabsible">
+                 <input id="collapsible2_2" class="toggle" type="checkbox">
+                <label for="collapsible2_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 class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Metabolic engineering
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Laying on the grass and desperately trying to fixate CO<sub>2</sub> myself.<br>
-              I keep you updated, if it works.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              An Eppendorf pencil. Totally worth it.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My Sanity and then my insanity and now i don't know anymore.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I didn't shaved my beard from July till wiki freeze.
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div class="sub" onclick="popup('rbn3')">
-          <div class="picture picture_michael"></div>
+           <div class="sub-header">
-           <div class="item-content">
+             <h1>
-             <p class="name">
+               F A C S
-               MICHAEL BURGIS
+             </h1>
-             </p>
+             <hr>
-             <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Strain engineering
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Mostly producing Vitamin D, doing sports and playing guitar.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              From time to time a headache, but all in all nothing too serious :)
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My favorite trousers :( RIP ✞
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I have once been called Dolly, cause my brothers look exactly the same as I do.
-            </p>
            </div>
-        </div>
+           <div class="sub-content">
-        <div class="item">
+             <div>
-           <div class="picture picture_jonas"></div>
+               Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-          <div class="item-content">
-            <p class="name">
-              JONAS FREUDIGMANN
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
              </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Strain engineering
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I use the sunlight to power my solar-bike:<br>
-              The photons excite me and bring me to a higher energy-level. Consequently I use these awesome biological
-              tools called ‘legs’ to move my bike!
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              The ability to peel sunflower seeds in my mouth - look ma no hands!
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Probably half of my friends, because I had no time for them all summer..<br>
-              If you read this, I am really sorry &lt;3
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              When listening to music while eating, I always chew to the rythm of the song.
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div id="rbn3" class="popup">
-          <div class="picture picture_maurice"></div>
+           <div class="popup-container">
-           <div class="item-content">
+             <div class="popup-header">
-             <p class="name">
+               <h1 class="title">Fluorescence-Activated Cell Sorting (FACS)</h1>
-               MAURICE MAGER
+               <button type="button" onclick="hide('rbn3')">X</button>
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-             <div class="columns">
+             <div class="popup-content" style="text-align: justify; text-align-last: justify;">
-               <div class="column is-3">
+              <p>
-                 <b>Field of Study:</b>
+                Abstract?
+              </p>
+              <br>
+              <br>
+               <div class="wrap-collabsible">
+                 <input id="collapsible3_1" class="toggle" type="checkbox">
+                <label for="collapsible3_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>
-               <div class="column item-column">
+              <br>
-                 Master’s student Molecular Biology and Cellular Biology
+               <div class="wrap-collabsible">
+                 <input id="collapsible3_2" class="toggle" ype="checkbox">
+                <label for="collapsible3_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 class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Benchmarking my own DNA repair systems.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              The eyesight to spot if I already put that one µl of
-              enzyme in all of my golden gate reaction mixes.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My trust in humanity as a whole.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              As a true german I eat around 2 kg of bread every week<br>
-              - 4 slices in the morning, 4 slices in the evening.
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div class="sub" onclick="popup('rbn4')">
-          <div class="picture picture_marian"></div>
+           <div class="sub-header">
-           <div class="item-content">
+             <h1>
-             <p class="name">
+               P A R T<br>
-               MARIAN KRÄMER
+               M E A S U R E M E N T
-            </p>
+             </h1>
-            <div class="columns">
+             <hr>
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-               My very white skin serves as a reflector to increase greenhouse effects.
-             </p>
-             <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Regularly Schrankdienst.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              You should rather ask what Maurice lost. ;)
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              Though many people think, I’m actually not a girl.<br>
-              Marian is a male name in Germany. :)
-            </p>
            </div>
-        </div>
+           <div class="sub-content">
-        <div class="item">
+             <div>
-           <div class="picture picture_paula"></div>
+               For our project it was indispensable to establish a measurement workflow that is not only applicable
-          <div class="item-content">
+               to UTEX 2973 and other cyanobacteria but also has a high throughput.
-            <p class="name">
-              PAULA
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Pharmacy
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Trying to produce sufficient amounts of vitamin D in ideal conditions:<br>
-+C/82+F, min. 1h spare time, enough UV-B radiation, in an outdoor pool with my peeps.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              An awesome wallpaper collection (including my alltime favorite: "The Honkler")<br>
-              - Thanks Schönas/Jonas
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Some of my eyesight :D had to get new glasses
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I like my bananas almost green and I have one each morning<br>
-              (like everyday...7 days a week, 365 days a year)
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div id="rbn4" class="popup">
-          <div class="picture picture_lars"></div>
+           <div class="popup-container">
-           <div class="item-content">
+             <div class="popup-header">
-            <p class="name">
+               <h1 class="title">Part Measurement</h1>
-              LARS BRÖKER
+               <button type="button" onclick="hide('rbn4')">X</button>
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-             <div class="columns">
+             <div class="popup-content">
-               <div class="column is-3">
+              <p>
-                 <b>Field of Study:</b>
+                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>
-               <div class="column item-column">
+              <br>
-                 Bachelor’s student Biology
+               <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 class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Just some biking and some more photography.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Voices in my head.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My circadian rhythm.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              My favourite animals are tardigrades.
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div class="sub" onclick="popup('rbn5')">
-          <div class="picture picture_wihan"></div>
+           <div class="sub-header">
-           <div class="item-content">
+             <h1>
-             <p class="name">
+               G R O W T H<br>
-               WIHAN ADI
+               C U R V E S
-            </p>
+             </h1>
-            <div class="columns">
+             <hr>
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student physics
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                DryLab
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-               Usually I like to jog, but after iGEM I might try to do photosynthesis instead.<br>
-              It seems more convenient.
-             </p>
-             <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Sunflower seeds addiction and some microbial friends, which are quite cool.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Sleepz and social lyfe.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              Sometimes I eat peanuts with their shells.
-            </p>
            </div>
-        </div>
+           <div class="sub-content">
-        <div class="item">
+             <div>
-           <div class="picture picture_annsophie"></div>
+               Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-          <div class="item-content">
-            <p class="name">
-              ANNSOPHIE WEBER
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Bioinformatics and Systems Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                DryLab
-              </div>
              </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              In my free time and in good weather I like to look after my plants on my balcony,
-              reading in the sun and preparing cool drinks with garden-fresh herbs.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              My first experiences in synthetic biology, more self esteem and a bunch of new friends.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              A lot of money, because of regularly ordering pizza or other food.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I'm afraid of sea cucumbers. Seriously, what are these things?!
-            </p>
            </div>
          </div>
-         <div class="item">
+         <div id="rbn5" class="popup">
-          <div class="picture picture_ting"></div>
+           <div class="popup-container">
-           <div class="item-content">
+             <div class="popup-header">
-            <p class="name">
+               <h1 class="title">Growth Curves</h1>
-              TING HIN WONG
+               <button type="button" onclick="hide('rbn5')">X</button>
-            </p>
-             <div class="columns">
-               <div class="column is-3">
-                <b>Age:</b>
-               </div>
-              <div class="column item-column">
-              </div>
              </div>
-             <div class="columns">
+             <div class="popup-content" style="text-align: justify; text-align-last: justify;">
-               <div class="column is-3">
+              <p>
-                 <b>Field of Study:</b>
+                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>
-              <div class="column item-column">
-                Master’s student Biotechnology/ Biopharmaceutical Technology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Metabolic engineering
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              On sunny days I like to enjoy refreshing drinks with his friends or just enjoying life at the
-              Schwanenteich in Gießen.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              I can fluently and by heart say the sentence:<br>
-              “We are working with <i>Synechococcus elongatus</i> UTEX 2973.”
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              “I never lose, either I win or I learn.”
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              My last name is “WONG” and not “WRONG”!
-            </p>
-          </div>
-        </div>
-        <div class="item">
-          <div class="picture picture_robin"></div>
-          <div class="item-content">
-            <p class="name">
-              ROBIN STEI
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Biotechnology/ Biopharmaceutical Technology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I most likely enjoy the sunlight while doing sports, especially playing soccer as a hobby or with my club.
                <br>
-               Besides that, I also like to relax in the garden and jump into the cold pool.
+               <div class="wrap-collabsible">
-            </p>
+                <input id="collapsible5_2" class="toggle" type="checkbox">
-            <b>What I gained in this iGEM year:</b><br>
+                <label for="collapsible5_2" class="lbl-toggle">
-            <p>
+                  <h3 class="title">Unterprojekt2</h3>
-              Still no beard...
+                </label>
-            </p>
+                <div class="collapsible-content">
-            <b>What I lost in this iGEM year:</b><br>
+                  <div class="content-inner" style="text-align: left; text-align-last: left;">
-            <p>
+                    <p>
-              Muscles...I’m not all the time a beanpole.
+                      Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-            </p>
+                    </p>
-            <b>A fun fact about me:</b><br>
+                  </div>
-            <p>
+                  <p>
-              People always say, that one leg is longer than the other. I never understood that, my leg is longer than
+                    One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications,
-              the other two...
+                    our team decided to redirect the metabolic flux of Synechococcus elongatus UTEX 2973 in order to synthesize add-value compounds
-            </p>
+                    with CO<sub>2</sub> and light as a resource. As a target, we thought about molecules, which also tackle one of the most important
-          </div>
+                    topic: the climate change.
-        </div>
+                  </p>
-        <div class="item">
+                  <br>
-          <div class="picture picture_chun"></div>
+                  <p>
-          <div class="item-content">
+                    Planes are considered to be one of the most environmentally damaging transport devices commonly used (<a style="padding: 0"
-            <p class="name">
+                      href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank">Borken-Kleefeld<i> et al.</i>, 2010</a>). A quick
-              CHUN-HO IP
+                    estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO<sub>2</sub> was released from the flights.
-            </p>
+                    To encounter this problem, we chose to set our focus on farnesene and limonene, which make up 90 % of the biojetfuel AMJ700t (50%
-            <div class="columns">
+                    limonene, 40% farnesene) from Amyris (<a style="padding: 0" href=" https://aem.asm.org/content/aem/81/10/3316.full.pdf"
-              <div class="column is-3">
+                      target="_blank">Brennan<i> et al.</i>, 2015</a>). This fuel has proven to be a suitable alternative to chemically produced, oil
-                <b>Age:</b>
+                    based <a style="padding: 0"
-              </div>
+                      href="
-              <div class="column item-column">
+https://investors.amyris.com/2012-06-19-Photo-Release-Azul-Brazilian-Airlines-Makes-Successful-Demonstration-Flight-With-Amyris-Renewable-Jet-Fuel-Produced-From-Sugarcane"
+                      target="_blank">jet fuels</a>
+                  </p>
+                  <br>
+                  <figure style="float:right; margin-left: 25px;">
+                    <img style="height: 500px; width: 750px;" src="https://2019.igem.org/File:T--Marburg--Metabolic--MEP-pathway.png"
+                      alt="Metabolic MEP Pathway">
+                    <figcaption style="max-width: 550px; text-align: center">
+                      Fig. 1- Overview of the MEP-Pathway. Enzymes are marked blue and Green. Limonene and farnesene Synthase are summarised as TPS.
+                      Modified after Lin et al 2016
+                    </figcaption>
+                  </figure>
+                  <p>
+                    Farnesene and limonene are both terpenoids, deriving from the so called 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (figure
+).
+                    The MEP pathway is a conserved pathway in bacteria and in photrophic organisms, it uses C3-bodies deriving from the Calvin-Cycle
+                    (directly as glyceraldehyde 3-phosphate and indirectly as pyruvate) as substrate. The resulting C5-bodiess, isopentenyl
+                    pyrophosphate (IPP) and it´s isomere dimethylallyl pyrophosphate (DAMPP) is used to build longer geranyl pyrophosphate (GPP) and
+                    Farnesyl pyrophosphate (FPP). GPP serves as the basis for a heterologous expressed limonene synthase to form D-Limonene, whereas
+                    FPP is a substrate for the plant derived Farnesene synthase.
+                    Because isoprenoids are interesting platform chemicals for various products, a lot of effort has been done to improve this pathway
+                    in cyanobacteria (<a style="padding: 0" href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i>
+                        et al.</i>, 2016</a>).
+                  </p>
+                  <br>
+                  <p>
+                    As a the first step we decided to heterologous express the limonene and the farnesene synthase to establish overexpression
+                    strains. We used the limonene synthase from <i>Lavandula angustifolia</i> and the Farnesen synthase from <i>Actinidia
+                      deliciosa</i> and codon optimised both enzymes.
+                    To redirect the flux into the MEP-Pathway we decided to overexpress the <i>E.Coli</i> proteins DXS, IDI and IspA. These targets
+                    were chosen on based previous results, which showed enhancement the production of amorpha-4,11-diene to 19.8 mg/L in PCC 7942
+                    without significant impairing the growth rate (<a style="padding: 0"
+                      href=" https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-016-0617-8" target="_blank">Choi<i> et
+                        al.</i>,2016</a>).
+                    Surprisingly, this suggest strong capacities of production in Cyanobacteria.
+                  </p>
+                  <br>
+                  <p>
+                    We chose the DXS, as many pathways are regulated on the first committing step. The IDI, would convert DMAPP to IPP, which is
+                    necessary to balance both pools, which is especially interesting for overproduction of Farnesene, where a ratio of 1:2 in favour
+                    of IPP is required. </p>
+                  <p>
+                    IspA can synthesise GPP and IPP to FPP, which would improve the efficiency in a farnesene production strain.
+                    To further enhance the efficiency of this pathway, we decided to mutate the DXS-residue 392 from a thyrosine (Y) to a
+                    phenylalanine (F). This would lead to a threefold increase in activity <i>in vitro</i> (<a style="padding: 0"
+                      href=" http://www.jbc.org/content/282/4/2676.long" target="_blank">Xiang<i> et al.</i>, 2016</a>).
+                    Also it would be advisable to remove the allosteric feedback regulation of the DXS (<a style="padding: 0"
+                      href=" https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161534” target=" _blank">Banerjee<i> et
+                        al.</i>,2016</a>).
+                  </p>
+                  <p>
+                    Removal of the end product is a key component in overproduction strains. Because farnesene and limonene are volatile, the
+                    extracellular diffusion rate is enough to prevent intracellular and potential toxic accumulation. On the other hand, a nontoxic
+                    overlay to catch the molecules is required, for example dodecan can be used. (<a style="padding: 0"
+                      href=" https://pubs.acs.org/doi/10.1021/acs.jafc.7b03625" target="_blank">Lee<i> et al.</i>, 2017</a>). An alternative supply
+                    with CO2 is also required, therefore we copied the system from Lee <i>et al. </i> and introduced a small tube with holes into the
+                    Medium.
+                  </p>
+                  <p>
+                    To balance the pathway we decided to use a weak promotor (<a style="padding: 0" href="http://parts.igem.org/Part:BBa_J23103"
+                      target="_blank">BBa_J23103</a>) for the ispA, as competition over FPP could lead to a heavy growth impact (<a style="padding: 0"
+                      href=" https://link.springer.com/article/10.1007/s00425-018-3047-y" target="_blank">Lin<i> et al.</i>, 2016</a>).
+                    As IDI is an Isomerase, only a comparably small amount of protein should be sufficient for a enhanced effect, so Promotor <a
+                      style="padding: 0" href="http://parts.igem.org/Part:BBa_J23110" target="_blank">BBa_J23110</a> was chosen. Because the DXS is
+                    the a potential bottle neck in Terpene Production, we chose the Promotor <a style="padding: 0"
+                      href="http://parts.igem.org/Part:BBa_J23111" target="_blank">BBa_J23111</a>.
+                  </p>
+                  <p>
+                    Sadly, we weren´t able to test our constructs <i>in vivo</i> and dropped this side project due to time reasons. As of now the
+                    submitted Parts for the limonene and the Farnesene synthase, which have been codon optimised for UTEX 2973 have been added to the
+                    registry ( <a style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228051" target="_blank"> K3228051</a> and <a
+                      style="padding: 0" href=" http://parts.igem.org/Part:BBa_K3228052" target="_blank"> K3228052</a>).
+                  </p>
+                 </div>
                </div>
              </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Chemistry
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I like to sunbathe and chill out with friends at the Lahn.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              I learned a lot about synthetic biology, which was a really nice experience.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Time to go on summer vacation … but now I’ll go to Boston :)
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              Sometimes I open the fridge for no reason.
-            </p>
            </div>
          </div>
-        <div class="item">
+       </section>
-          <div class="picture picture_johanna"></div>
+     </div>
-          <div class="item-content">
-            <p class="name">
-              JOHANNA WALLBAUM
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Chemistry
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I enjoy the sunlight the most when I'm hanging out with my friends on the Lahnwiesen.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Knowledge of synthetic biology.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              A few tears.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I know "I love you" in 18 languages.
-            </p>
-          </div>
-        </div>
-        <div class="item">
-          <div class="picture picture_joana"></div>
-          <div class="item-content">
-            <p class="name">
-              JOANA ESSLEN
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              I use the sunlight in my leisure time to dream about lying on the beach in Portugal with a cocktail.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              An addiction to Sterillium classic pure Händedesinfektionsmittel.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              My roommate-fish Steve I had for not being alone at home. He died probably because I never had
-              time to care for him.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              WAS? WAS? WAS? - I’m kind of nosey.
-            </p>
-          </div>
-        </div>
-        <div class="item">
-          <div class="picture picture_jana"></div>
-          <div class="item-content">
-            <p class="name">
-              JANA SIPPEL
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Bachelor’s student Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Toolbox
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Meet friends and enjoy the nature
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              New working experience and new friends.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Billions of green friends.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I want a cow which is able to do photosynthesis.
-            </p>
-          </div>
-        </div>
-        <div class="item">
-          <div class="picture picture_julia"></div>
-          <div class="item-content">
-            <p class="name">
-              JULIA SEUFER
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Master’s student Molecular and Cellular Biology
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Metabolic engineering
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Macrophotography and determination of Hymenoptera. Mushroom hunting, hiking.
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              Appreciation for reliable colleagues.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              I haven't lost anything.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              Nothing gets me quite as excited as the endless possibilities of discovery in a puddle of bacterial
-              mystery goo :D
-            </p>
-          </div>
-        </div>
-        <div class="item">
-          <div class="picture picture_erik"></div>
-          <div class="item-content">
-            <p class="name">
-              ERIK BENDER
-            </p>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Age:</b>
-              </div>
-              <div class="column item-column">
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Field of Study:</b>
-              </div>
-              <div class="column item-column">
-                Software Development
-              </div>
-            </div>
-            <div class="columns">
-              <div class="column is-3">
-                <b>Subgroup:</b>
-              </div>
-              <div class="column item-column">
-                Wiki Wizard 🧙&zwj;♂️
-              </div>
-            </div>
-            <b>How I 'use' the sunlight in my leisure time:</b><br>
-            <p>
-              Mostly trying to keep it out of my room ^^
-            </p>
-            <b>What I gained in this iGEM year:</b><br>
-            <p>
-              New friends and the feeling of being able to make a difference.
-            </p>
-            <b>What I lost in this iGEM year:</b><br>
-            <p>
-              Just some free time.
-            </p>
-            <b>A fun fact about me:</b><br>
-            <p>
-              I came to iGEM by chance and stayed.
-            </p>
-          </div>
-        </div>
-        <hr>
-        <h1 class="title">Principal Investigators</h1>
-        <ul>
-          <li class="name" style="margin-bottom: 1em;">
-            Prof. Dr. Lars-Oliver Essen
-          </li>
-          <li class="name">Prof. Dr. Lars Matthias Voll</li>
-        </ul>
-       </div>
-     </section>
    </div>
 </html>
 {{Marburg/footer}}

Difference between revisions of "Team:Marburg/test"

Latest revision as of 02:41, 14 December 2019

G I A N T J A M B O R E E

Storytelling:

L I G H T M E A S U R E M E N T

Light Measurement

R E P O R T E R S

Reporter

Unterprojekt1

Unterprojekt2

F A C S

Fluorescence-Activated Cell Sorting (FACS)

Unterprojekt1

Unterprojekt2

P A R T M E A S U R E M E N T

Part Measurement

Experimental Procedure

Data analysis and evaluation

G R O W T H C U R V E S

Growth Curves

Unterprojekt1

Unterprojekt2

G I A N T
J A M B O R E E

L I G H T
M E A S U R E M E N T

P A R T
M E A S U R E M E N T

G R O W T H
C U R V E S