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

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R E P O R T E R S

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F A C S

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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

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Abstract?

Unterprojekt1

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Unterprojekt2

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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}}
 <html>
-<body style="overflow: auto; max-height: 100vh;">
+  <div>
-    <center>
+    <div class="box-dark">
-        <h1 class="title">Measurement</h1>
-     </center>
+     <h1 class="heading" style="
-     <br>
+    line-height: 1;
-    <br>
+">
-    <p>
-        <h1 class="subtitle">Amplifying new standards in measurement</h1><br>
+        G I A N T<br> J A M B O R E E
-         <p style="text-align: justify;">
+      </h1>
-             <br>
+      <hr class="line">
-             Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
+      <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo">
-             <br>
+     </div>
-             <br>
+     <div style="margin-top: 10vh;">
+      <section class="section">
+      <div style="display: flex; flex-direction: row;">
+<p style="margin-bottom: 1em;">
+         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.
+</p>
+                        <figure style="text-align: center; margin-left: 25px; max-width: 42000px">
+                          <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>
+                        </div>
+<p style="margin-bottom: 1em;">
+<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>
+<br>
+<br>
+<p style="margin-bottom: 1em;">
+<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>
+ <p style="margin-bottom: 1em;">
+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!
+        </p>
+<br><br><br>
+        <figure style="float:right; margin-left: 25px; text-align: center;">
+          <img style="height: 500px; mix-blend-mode: multiply;" src="https://static.igem.org/mediawiki/2019/7/7d/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>
-        <h1 class="subtitle">Storytelling:</h1><br>
-         <br>
-         <p style="text-align: justify;">
-             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.
-             <br>
+<figure style="float:right; margin-left: 25px; text-align: center;">
-             <br>
+          <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;">
+         <p style="margin-bottom: 1em;">
+         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.
+</p>
+      </section>
+      <section class="section">
+        <article>
+          <h1 class="title">Storytelling:</h1>
+          <p style="text-align: justify; margin-bottom: 1em;">
+             We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX
+, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us
+            in front of many problems and questions. Especially the usage of different media, light conditions and other
+             cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers.
+            Many of these problems are reasoned in the ongoing optimization and development of methods and instruments.
+             Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic
+             microbiology and necessary to compare results with other scientists and reproduce their data.
+          </p>
+          <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">
+              Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus
+              UTEX
+Absorption spectra.
+            </figcaption>
+          </figure>
+          <p style="text-align: justify; margin-bottom: 1em;">
              While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to
              show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore
-             we
+             we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
-            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
-             light
+             already known FACS measurement method (?).
-            measurement, evaluated different reporters???, established a measurement method and compared it to a already
+          </p>
-             known FACS measurement method (?).
+          <p style="text-align: justify; margin-bottom: 1em;">
-            <br>
-            <br>
              At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle].
-             So we
+             So we had to measure the light conditions in our incubators and while doing this simple task the first
-            had to measure the light conditions in our incubators and while doing this simple task the first part of
+             part of standardization began. We discovered that nearly every paper? is using different methods to measure
-             standardization began. We discovered that nearly every paper? is using different methods to measure their
+             their light conditions and that it is a really complex and important procedure. So we got in contact with
-             light
+            cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following
-            conditions and that it is a really complex and important procedure. So we got in contact with cyano and
+            popup we show different ways of measurement, their (dis-)advantages and different results depending on the
-            light
+             measuring instrument.<br>
-            measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show
+             Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed.
-            different ways of measurement, their (dis-)advantages and different results depending on the measuring
+             In literature and while talking with different experts (IHP), we recognized that small deviations of these
-             instrument.<br>
-             Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In
-             literature and while talking with different experts (IHP), we recognized that small deviations of these
              parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as
-             a
+             a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters
-            new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters
+             that lead to the fastest growth speed.<br>
-             that
-            lead to the fastest growth speed.<br>
              Another aspect was measuring the expression and characterize our part. Different possibilities were
-             discussed
+             discussed and after testing them we decided on two methods in our project (plate reader and FACs). One
-            and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to
+            approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate
-            measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to
+            readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br>
-            standard equipment of every lab nowadays, and could deliver easy reproducible results.<br>
              The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In
              contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its
-             own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not every
+             own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not
-             laboratory
+             every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database
-            posses a FACs/device. So in the end we would like to offer a two method analyzed database from our
+            from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the
-            crontructs
+            difference in measurement methods.<br>
-            for iGEM teams and research groups, who do not have access to a FACS and show the difference in measurement
-            methods.<br>
              At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973
-             and
+             and make a contribution to the cyano community by establishing essential/fixed standards in measurement.
-            make a contribution to the cyano community by establishing essential/fixed standards in measurement.
+          </p>
-            <br>
+         </article>
-         </p>
+      </section>
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+              L I G H T<br>
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+              M E A S U R E M E N T
-                        <h2>Light measurement</h2>
+            </h1>
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+                Abstract?
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+                    Unterprojekt2
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+            <hr>
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-                        <h2>Reporters</h2>
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-                <br>
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+            <hr>
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+          </div>
-                        <h2>Fluorescence-Activated Cell Sorting (FACS)</h2>
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+            </div>
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-                <h1>Fluorescence-Activated Cell Sorting (FACS)</h1>
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+                    </p>
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+                  </div>
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+              </div>
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+          </div>
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-                    <div class="content">
+            <hr>
-                        <h2>Part Measurement</h2>
+          </div>
-                        <p>
+          <div class="sub-content">
-                            For our project it was indispensable to establish a measurement workflow that is not only
+            <div>
-                            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
-                        </p>
+              to UTEX 2973 and other cyanobacteria but also has a high throughput.
-                    </div>
+            </div>
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+          <div class="popup-container">
-        <div class="popup-container">
              <div class="popup-header">
-                <h1>Part Measurement</h1>
+              <h1 class="title">Part Measurement</h1>
-                <button type="button" onclick="hide('rbn4')">X</button>
+              <button type="button" onclick="hide('rbn4')">X</button>
              </div>
-             <div class="popup-content" style="text-align: justify; text-align-last: justify;">
+             <div class="popup-content">
-                <p>
+              <p>
-                    For our project it was indispensable to establish a measurement workflow that is not only applicable
+                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.
+                to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg
-                    While we worked on our Marburg Collection 2.0 with XXX parts we came to the conclusion it is also
+                Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement
-                    necessary to develop a measurement method that suites such a large collection. Therefore we
+                method that suites such a large collection. Therefore we elaborated different workflows - containing
-                    elaborated different workflows - containing different cultivation vessels and parameters - and
+                different cultivation vessels and parameters - and revised them after evaluating the results. In the end
-                    revised them after evaluating the results. In the end we were able to establish a workflow specially
+                we were able to establish a workflow specially designed for our methods to cultivate and characterize
-                    designed for our methods to cultivate and characterize the parts from our Marburg Collection 2.0,
+                the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX 2973.
-                    that is tailored to <i>Synechococcus elongatus</i> UTEX 2973.
+              </p>
-                </p>
+              <div class="wrap-collabsible">
-                <br>
+                <input id="collapsible4_1" class="toggle" type="checkbox">
-                <br>
+                <label for="collapsible4_1" class="lbl-toggle">
-                <div class="wrap-collabsible">
+                  <h3 class="title" style="text-align: left; text-align-last: left;">
-                    <input id="collapsible4_1" class="toggle" type="checkbox">
+                    Experimental Procedure
-                    <label for="collapsible4_1" class="lbl-toggle">
+                  </h3>
-                        <h3 class="title" style="text-align: left; text-align-last: left;">Experimental Procedure</h3>
+                </label>
-                    </label>
+                <div class="collapsible-content">
-                    <div class="collapsible-content">
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-                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+                    <p>
-                            <p>
+                      The results of our part characterization were obtained by fluorescence and luminescence
-                                The results of our part characterization were obtained by fluorescence and luminescence
+                      measurements (of what?). But before the party could be measured we had to
-                                measurements (of what?). But before the party could be measured we had to elaborate a
+                      elaborate a cultivating and measuring workflow.<br>
-                                cultivating and measuring workflow.
+                      For the cultivating workflow we tested different well plate formats and growing parameters for the
-                                <br>
+                      best growing conditions. It was logistically the best way to cultivate and measure the parts in
-                                For the cultivating workflow we tested different well plate
+                      well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space
-                                formats and growing parameters for the best growing conditions. It was logistically the
+                      in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
-                                best way to cultivate and measure the parts in well plates, because the Marburg
+                        elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small
-                                Collection 2.0 comprises xxx parts and we were limited in space in our incubator.
+                      clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of
-                                Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
+                      the incubator was limited whereas cultures in flasks had to be incubated at the same time and
-                                    elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures
+                      these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating
-                                showed small clouds of cells formed by inappropriate movement of media in the wells. In
+                      flasks and well-plates in the same incubator. After revising the workflow over and over we came to
-                                addition, the rpm of the incubator was limited whereas cultures in flasks had to be
+                      the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates
-                                incubated at the same time and these threatened to fall over at high rpm. At 130 rpm we
+                      because there was enough movement in the wells to prevent the cells from forming a pellet/cloud.
-                                found a compromise between cultivating flasks and well-plates in the same incubator.
+                      Further it was necessary to use transparent wells to ensure every well with similar ight
-                                After revising the workflow over and over we came to the conclusion, that it is
+                      conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?)
-                                favorable to cultivate the UTEX 2973 in transparent 24-well-plates because there was
+                      growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an
-                                enough movement in the wells to prevent the cells from forming a pellet/cloud. Further
+                      important role in cultivation of well plates cause the realtive small volumes and high surfaces
-                                it was necessary to use transparent wells to ensure every well with similar light
+                      (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is
-                                conditions. Concerning of light conditions, we evaluated that the cells showed good
+                      essential to know the volume in the wells for measuring in the plate reader. Therefore we compared
-                                (prosperous?) growth in the wells at low-light conditions (around 500 µE). The
+                      different seals for the well plates and in the end we came to the conclusion that using a
-                                evaporation of medium plays an important role in cultivation of well plates cause the
+                      semipermeable foil is the best solution. The evaporation could be minimalized and the cells were
-                                realtive small volumes and high surfaces (ich glaub die flache ist eher klein, aber vllt
+                      able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to
-                                wegen der Temperatur und Zeit?). Further it is essential to know the volume in the wells
+                      cultivate the cells for 2-3 days without losing significant amounts of medium.
-                                for measuring in the plate reader. Therefore we compared different seals for the well
+                      <br>
-                                plates and in the end we came to the conclusion that using a semipermeable foil is the
+                      <br>
-                                best solution. The evaporation could be minimalized and the cells were able to get
+                      <center>xxxx
-                                enough CO2 because air transfer was provide/permit. By using a foil it was possible to
+                        Fig x.:Schema vom Workflow</center>
-                                cultivate the cells for 2-3 days without losing significant amounts of medium.
+                      <br>
-                                <br>
+                      As described before we used the following workflow as shown in fig. XX to cultivate and measure
-                                <br>
+                      our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the
-                                <center>xxxx
+                      end of the triparental conjugation (LINK). For every part we picked 3 different colonies and
-                                    Fig x.:Schema vom Workflow</center>
+                      inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates
-                                <br>
+                      we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to
-                                As described before we used the following workflow as shown in fig. XX to cultivate and
+                      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
-                                measure our parts. The cultivation started by picking colonies from BG11-agar-plates
+                      A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was
-                                that were used at the end of the triparental conjugation (LINK). For every part we
+                      inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while
-                                picked 3 different colonies and inoculated them in 1.0 mL BG11-media with 0.5 µl
+                      evaluating the results (that will be used as a blank while ...). When all the cultures in the
-                                Spectinomycin. Thus in the first 24-well-plates we could inoculate 8 different parts
+                      second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same
-                                with 3 biological parallels. When the cultures grew to OD<sub>730</sub>=0.6-0.8 they
+                      well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating
-                                were
+                      technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When
-                                inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells A1-3 (part 1) and A4-6 (part
+                      the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred
-) of
+                      into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three
-                                another 24-well-plate. At the same time the Well B6 was inoculated with 1.0 mL of a
+                      times. Following this workflow we were able to measure three biological parallels and
-                                OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while evaluating the results
+                      two technical parallels for every biological parallel. It enabled us to have a good statistical
-                                (that will
+                      database and gives our results a stronger meaning/significance. While working with this workflow
-                                be used as a blank while ...). When all the cultures in the second 24-well-plate reached
+                      it was essential to keep the cultures in their exponential phase because it would significantly
-                                OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same well-plate. It was done
+                      speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es
-                                by
+                      erst gar keine lag phase gibt).<br>
-                                inoculating the wells A1-3 into the wells C1-3 and D1-3 creating technical parallels of
+                      Concerning the measurement part we decided to transfer the cultures into black/white luminescence
-                                the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When the wells
+                      is measured in white ones. We measured in 96-well-plates because it enabled us to measure every
-                                C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred
+                      part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure
-                                into a
+                      eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for
--well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three
+                      measurement)<br>
-                                times. Following this workflow we were able to measure three biological parallels and
+                      <br>
-                                two technical parallels for every biological parallel. It enabled us to have a good
+                      <b>Fluorescence measurement:</b><br>
-                                statistical database and gives our results a stronger meaning/significance. While
+                      After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured.
-                                working with this workflow it was essential to keep the cultures in their exponential
+                      More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP
-                                phase because it would significantly speed up the growth by reducing the lag-phase to an
+                      fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the
-                                absolute minimum (oder lieber sagen dass es erst gar keine lag phase gibt).
+                      considered part. For measurement we created a program that measured the OD<sub>730</sub> and the
-                                <br>
+                      fluorescence of the wells.<br>
-                                Concerning the measurement part we decided to transfer the cultures into black/white
+                      <br>
--well-plates. While fluorescence is measured in black well-plates the luminescence is
+                      <center>fig XX (screenshot des messprogams)</center>
-                                measured in white ones. We measured in 96-well-plates because it enabled us to measure
+                      <br>
-                                every part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further
+                      In order to measure the OD in each well we determined the absorption at 730 nm. Further we
-                                we could measure eight (?) parts in only one plate. (four 24-well-plates lead into one
+                      measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the
--well-plate for measurement)
+                      border of the well showed consistent results with small standard deviations (fig. XX). We used the
-                                <br>
+                      same settings of the multiple measurement for the fluorescence measurement. While using sYFP as
-                                <br>
+                      signal for our part measurement we have set the emission wavelength to 515 nm and the excitation
-                                <b>Fluorescence measurement:</b><br>
+                      wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database
-                                After transfering the cultures into the 96-well-plate the fluorescence of the parts was
+                      verlinken/als quelle?)<br>
-                                measured. More precisely, the activity of the parts was determined by the expression of
+                      <br>
-                                the sYFP. The sYFP fluorescence served as an indicator and the sequence for the sYFP was
+                      <b>Fluorescence-Activated Cell Sorting (FACS):</b><br>
-                                in the same cassette as the considered part.
+                      short abstract and link to the FACS-text of the measurement
-                                For measurement we created a program that measured the OD<sub>730</sub> and the
+                      <br>
-                                fluorescence of the
+                      <br>
-                                wells.
+                      <b>Luminescence Measurement</b><br>
-                                <br>
+                      <br>
-                                <br>
+                      text
-                                <center>fig XX (screenshot des messprogams)</center>
+                    </p>
-                                <br>
+                  </div>
-                                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 1350
-                                nm to the border of the well showed consistent results with small standard deviations
-                                (fig. XX).
-                                We used the same settings of the multiple measurement for the fluorescence measurement.
-                                While using sYFP as signal for our part measurement we have set the emission wavelength
-                                to 515 nm and the excitation wavelength to 527 nm, fitting the exact wavelengths of the
-                                sYFP shown in XX (Database verlinken/als quelle?)
-                                <br>
-                                <br>
-                                <b>Fluorescence-Activated Cell Sorting (FACS):</b><br>
-                                short abstract and link to the FACS-text of the measurement
-                                <br>
-                                <br>
-                                <b>Luminescence Measurement</b><br>
-                                <br>
-                                text
-                            </p>
-                        </div>
-                    </div>
                  </div>
-                <br>
+              </div>
-                <div class="wrap-collabsible">
+              <br>
-                    <input id="collapsible4_2" class="toggle" type="checkbox">
+              <div class="wrap-collabsible">
-                    <label for="collapsible4_2" class="lbl-toggle">
+                <input id="collapsible4_2" class="toggle" type="checkbox">
-                        <h3 class="title" style="text-align: left; text-align-last: left;">Data analysis and evaluation
+                <label for="collapsible4_2" class="lbl-toggle">
-                        </h3>
+                  <h3 class="title" tyle="text-align: left; text-align-last: left;">Data analysis and evaluation
-                    </label>
+                  </h3>
-                    <div class="collapsible-content">
+                </label>
-                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+                <div class="collapsible-content">
-                            <p>
+                  <div class="content-inner" style="text-align: left; text-align-last: left;">
-                                kein plan was man hier schreiben soll zum jetzigen standpunkt...
+                    <p>
-                                For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11)
+                      kein plan was man hier schreiben soll zum jetzigen standpunkt...
-                                and for the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
+                      For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for
-                                <br>
+                      the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
-                                Auswertung, Daten und Grafen darstellen?
+                      <br>
-                            </p>
+                      Auswertung, Daten und Grafen darstellen?
-                        </div>
+                    </p>
-                    </div>
+                  </div>
                  </div>
+              </div>
              </div>
+          </div>
          </div>
-    </div>
+         <div class="sub" onclick="popup('rbn5')">
-    <br>
+          <div class="sub-header">
-    <div class="container">
+            <h1>
-         <div class="box" style="cursor: pointer;" onclick="popup('rbn4')">
+              G R O W T H<br>
-            <article class="media">
+              C U R V E S
-                <div class="media-content">
+            </h1>
-                    <div class="content">
+            <hr>
-                        <h2>Growth Curves</h2>
+          </div>
-                        <p>
+          <div class="sub-content">
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+            <div>
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-                    </div>
+            </div>
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-            </article>
          </div>
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+          <div class="popup-container">
-        <div class="popup-container">
              <div class="popup-header">
-                <h1>Growth Curves</h1>
+              <h1 class="title">Growth Curves</h1>
-                <button type="button" onclick="hide('rbn4')">X</button>
+              <button type="button" onclick="hide('rbn5')">X</button>
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              <div class="popup-content" style="text-align: justify; text-align-last: justify;">
-                <p>
+              <p>
-                    Abstract?
+                Abstract?
-                </p>
+              </p>
-                <br>
+              <br>
-                <br>
+              <br>
-                <div class="wrap-collabsible">
+              <div class="wrap-collabsible">
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+                <label for="collapsible5_1" class="lbl-toggle">
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+                  <h3 class="title">Unterprojekt1</h3>
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+                </label>
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+                <div class="collapsible-content">
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+                  <div class="content-inner" style="text-align: left; text-align-last: left;">
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+                    </p>
-                        </div>
+                  </div>
-                    </div>
                  </div>
-                <br>
+              </div>
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+              <br>
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-                        <h3 class="title">Unterprojekt2</h3>
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-                    </label>
+                  <h3 class="title">Unterprojekt2</h3>
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+                </label>
-                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+                <div class="collapsible-content">
-                            <p>
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+                    <p>
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+                      Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
-                        </div>
+                    </p>
-                     </div>
+                  </div>
+                  <p>
+                     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 CO<sub>2</sub> and light as a resource. As a target, we thought about molecules, which also tackle one of the most important
+                    topic: the climate change.
+                  </p>
+                  <br>
+                  <p>
+                    Planes are considered to be one of the most environmentally damaging transport devices commonly used (<a style="padding: 0"
+                      href=" https://pubs.acs.org/doi/full/10.1021/es9039693" target="_blank">Borken-Kleefeld<i> et al.</i>, 2010</a>). A quick
+                    estimation suggests, yet only for the Giant Jamboree in Boston 2018, 14.000.000 tons CO<sub>2</sub> 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 (<a style="padding: 0" href=" https://aem.asm.org/content/aem/81/10/3316.full.pdf"
+                      target="_blank">Brennan<i> et al.</i>, 2015</a>). This fuel has proven to be a suitable alternative to chemically produced, oil
+                    based <a style="padding: 0"
+                      href="
+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>
          </div>
+      </section>
      </div>
-</body>
+  </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

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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