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| <div> | | <div> |
| <div class="box-dark"> | | <div class="box-dark"> |
− | <h1 class="heading">
| + | |
− | M E A S U R E M E N T | + | <h1 class="heading" style=" |
| + | line-height: 1; |
| + | "> |
| + | |
| + | G I A N T<br> J A M B O R E E |
| </h1> | | </h1> |
| <hr class="line"> | | <hr class="line"> |
− | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" | + | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo"> |
− | class="logo"
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− | alt="Syntex Logo">
| + | |
| </div> | | </div> |
| <div style="margin-top: 10vh;"> | | <div style="margin-top: 10vh;"> |
| <section class="section"> | | <section class="section"> |
− | <h1 class="title">Amplifying new standards in measurement</h1>
| + | <div style="display: flex; flex-direction: row;"> |
− | <p style="text-align: justify;"> | + | |
− | Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS) | + | <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> | | </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>, |
| + | 2016</a>). |
| + | </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;"> |
| + | |
| + | <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> |
| <section class="section"> | | <section class="section"> |
− | <h1 class="title">Storytelling:</h1> | + | <article> |
− | <p style="text-align: justify;">
| + | <h1 class="title">Storytelling:</h1> |
− | We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in
| + | We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX |
− | front of many problems and questions. Especially the usage of different media, light conditions and other
| + | 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us |
− | cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers.
| + | in front of many problems and questions. Especially the usage of different media, light conditions and other |
− | Many of these problems are reasoned in the ongoing optimization and development of methods and instruments.
| + | cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. |
− | Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic
| + | Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. |
− | microbiology and necessary to compare results with other scientists and reproduce their data.
| + | Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic |
− | </p>
| + | microbiology and necessary to compare results with other scientists and reproduce their data. |
− | <p>
| + | </p> |
− | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to | + | <figure style="float:right; margin-left: 25px;"> |
− | show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore
| + | <img style="height: 719px; width: 1001px;" src="https://2019.igem.org/File:T--Marburg--Metabolic--MEP-pathway.png" alt="asas"> |
− | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
| + | <figcaption style="max-width: 550px; text-align: center"> |
− | light measurement, evaluated different reporters???, established a measurement method and compared it to a
| + | Fig.1 - Comparison of NanoLuc and teLuc Luminescence Spectra in comparison with Synechococcus elongatus |
− | already known FACS measurement method (?).
| + | UTEX |
− | </p>
| + | 2973 Absorption spectra. |
− | <p>
| + | </figcaption> |
− | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle].
| + | </figure> |
− | So we had to measure the light conditions in our incubators and while doing this simple task the first
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | part of standardization began. We discovered that nearly every paper? is using different methods to measure
| + | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to |
− | their light conditions and that it is a really complex and important procedure. So we got in contact with
| + | show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore |
− | cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following
| + | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in |
− | popup we show different ways of measurement, their (dis-)advantages and different results depending on the
| + | light measurement, evaluated different reporters???, established a measurement method and compared it to a |
− | measuring instrument.<br>
| + | already known FACS measurement method (?). |
− | Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed.
| + | </p> |
− | In literature and while talking with different experts (IHP), we recognized that small deviations of these
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as
| + | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. |
− | a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters
| + | So we had to measure the light conditions in our incubators and while doing this simple task the first |
− | that lead to the fastest growth speed.<br>
| + | part of standardization began. We discovered that nearly every paper? is using different methods to measure |
− | Another aspect was measuring the expression and characterize our part. Different possibilities were discussed
| + | their light conditions and that it is a really complex and important procedure. So we got in contact with |
− | and after testing them we decided on two methods in our project (plate reader and FACs). One
| + | cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following |
− | approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate
| + | popup we show different ways of measurement, their (dis-)advantages and different results depending on the |
− | readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br>
| + | measuring instrument.<br> |
− | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In
| + | Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. |
− | contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its
| + | In literature and while talking with different experts (IHP), we recognized that small deviations of these |
− | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not
| + | parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as |
− | every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database
| + | a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters |
− | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the
| + | that lead to the fastest growth speed.<br> |
− | difference in measurement methods.<br>
| + | Another aspect was measuring the expression and characterize our part. Different possibilities were |
− | At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973
| + | discussed and after testing them we decided on two methods in our project (plate reader and FACs). One |
− | and make a contribution to the cyano community by establishing essential/fixed standards in measurement.
| + | approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate |
− | </p>
| + | readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br> |
| + | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In |
| + | contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its |
| + | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not |
| + | every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database |
| + | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the |
| + | difference in measurement methods.<br> |
| + | At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 |
| + | and make a contribution to the cyano community by establishing essential/fixed standards in measurement. |
| + | </p> |
| + | </article> |
| </section> | | </section> |
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| + | <h3 class="title">Unterprojekt1</h3> |
− | style="text-align: left; text-align-last: left;">
| + | </label> |
− | <p>
| + | <div class="collapsible-content"> |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
− | ist.
| + | <p> |
− | </p>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| + | </p> |
| + | </div> |
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− | </div>
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− | <br>
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− | type="checkbox">
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− | <label for="collapsible2_2"
| + | <div class="collapsible-content"> |
− | class="lbl-toggle">
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
− | <h3 class="title">Unterprojekt2</h3>
| + | <p> |
− | </label>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | <div class="collapsible-content">
| + | </p> |
− | <div class="content-inner"
| + | </div> |
− | style="text-align: left; text-align-last: left;">
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− | <p>
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− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig
| + | |
− | ist.
| + | |
− | </p>
| + | |
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| </div> | | </div> |
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− | </div>
| + | <div class="sub" onclick="popup('rbn3')"> |
− | <br>
| + | <div class="sub-header"> |
− | <div class="container">
| + | <h1> |
− | <div class="box" | + | F A C S |
− | style="cursor: pointer;"
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− | onclick="popup('rbn3')">
| + | <hr> |
− | <article class="media"> | + | </div> |
− | <div class="media-content"> | + | <div class="sub-content"> |
− | <div class="content"> | + | <div> |
− | <h2>Fluorescence-Activated Cell Sorting (FACS)</h2>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | <p>
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| + | <h1 class="title">Fluorescence-Activated Cell Sorting (FACS)</h1> |
− | <div class="popup-header">
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− | <h1>Fluorescence-Activated Cell Sorting (FACS)</h1>
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− | <button type="button"
| + | <div class="popup-content" style="text-align: justify; text-align-last: justify;"> |
− | onclick="hide('rbn3')">X</button>
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− | </div>
| + | Abstract? |
− | <div class="popup-content"
| + | </p> |
− | style="text-align: justify; text-align-last: justify;">
| + | <br> |
− | <p>
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− | Abstract?
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− | <br>
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− | <label for="collapsible3_1"
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | class="lbl-toggle">
| + | </p> |
− | <h3 class="title">Unterprojekt1</h3>
| + | </div> |
− | </label>
| + | |
− | <div class="collapsible-content">
| + | |
− | <div class="content-inner"
| + | |
− | style="text-align: left; text-align-last: left;">
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− | <p>
| + | |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig
| + | |
− | ist.
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− | </p>
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− | </div>
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− | <br>
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− | <div class="wrap-collabsible">
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− | class="lbl-toggle">
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− | <h3 class="title">Unterprojekt2</h3>
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− | </label>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | <div class="collapsible-content">
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− | <div class="content-inner"
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− | style="text-align: left; text-align-last: left;">
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− | <p>
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− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig
| + | |
− | ist.
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− | </div>
| + | <div class="sub" onclick="popup('rbn4')"> |
− | <br>
| + | <div class="sub-header"> |
− | <div class="container">
| + | <h1> |
− | <div class="box" | + | P A R T<br> |
− | style="cursor: pointer;"
| + | M E A S U R E M E N T |
− | onclick="popup('rbn4')">
| + | </h1> |
− | <article class="media"> | + | <hr> |
− | <div class="media-content"> | + | </div> |
− | <div class="content"> | + | <div class="sub-content"> |
− | <h2>Part Measurement</h2>
| + | <div> |
− | <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
| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. |
− | only
| + | |
− | applicable to UTEX 2973 and other cyanobacteria but also has a high throughput.
| + | |
− | </p>
| + | |
− | </div>
| + | |
| </div> | | </div> |
− | </article> | + | </div> |
| </div> | | </div> |
− | </div>
| + | <div id="rbn4" class="popup"> |
− | <div id="rbn4"
| + | <div class="popup-container"> |
− | class="popup">
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− | <div class="popup-container">
| + | <h1 class="title">Part Measurement</h1> |
− | <div class="popup-header">
| + | <button type="button" onclick="hide('rbn4')">X</button> |
− | <h1>Part Measurement</h1>
| + | </div> |
− | <button type="button"
| + | <div class="popup-content"> |
− | onclick="hide('rbn4')">X</button>
| + | <p> |
− | </div>
| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
− | <div class="popup-content"
| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg |
− | style="text-align: justify; text-align-last: justify;">
| + | Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement |
− | <p>
| + | method that suites such a large collection. Therefore we elaborated different workflows - containing |
− | For our project it was indispensable to establish a measurement workflow that is not only
| + | different cultivation vessels and parameters - and revised them after evaluating the results. In the end |
− | applicable
| + | we were able to establish a workflow specially designed for our methods to cultivate and characterize |
− | to UTEX 2973 and other cyanobacteria but also has a high throughput.
| + | the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX 2973. |
− | While we worked on our Marburg Collection 2.0 with XXX parts we came to the conclusion it is
| + | </p> |
− | also
| + | <div class="wrap-collabsible"> |
− | necessary to develop a measurement method that suites such a large collection. Therefore we
| + | <input id="collapsible4_1" class="toggle" type="checkbox"> |
− | elaborated different workflows - containing different cultivation vessels and parameters -
| + | <label for="collapsible4_1" class="lbl-toggle"> |
− | and
| + | <h3 class="title" style="text-align: left; text-align-last: left;"> |
− | revised them after evaluating the results. In the end we were able to establish a workflow
| + | Experimental Procedure |
− | specially
| + | </h3> |
− | designed for our methods to cultivate and characterize the parts from our Marburg Collection
| + | </label> |
− | 2.0,
| + | <div class="collapsible-content"> |
− | that is tailored to <i>Synechococcus elongatus</i> UTEX 2973.
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
− | </p>
| + | <p> |
− | <br>
| + | The results of our part characterization were obtained by fluorescence and luminescence |
− | <br>
| + | measurements (of what?). But before the party could be measured we had to |
− | <div class="wrap-collabsible">
| + | elaborate a cultivating and measuring workflow.<br> |
− | <input id="collapsible4_1"
| + | For the cultivating workflow we tested different well plate formats and growing parameters for the |
− | class="toggle"
| + | best growing conditions. It was logistically the best way to cultivate and measure the parts in |
− | type="checkbox">
| + | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space |
− | <label for="collapsible4_1"
| + | in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus |
− | class="lbl-toggle">
| + | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small |
− | <h3 class="title"
| + | clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of |
− | style="text-align: left; text-align-last: left;">Experimental Procedure</h3>
| + | the incubator was limited whereas cultures in flasks had to be incubated at the same time and |
− | </label>
| + | these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating |
− | <div class="collapsible-content">
| + | flasks and well-plates in the same incubator. After revising the workflow over and over we came to |
− | <div class="content-inner"
| + | the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates |
− | style="text-align: left; text-align-last: left;">
| + | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. |
− | <p>
| + | Further it was necessary to use transparent wells to ensure every well with similar ight |
− | The results of our part characterization were obtained by fluorescence and
| + | conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) |
− | luminescence
| + | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an |
− | measurements (of what?). But before the party could be measured we had to
| + | important role in cultivation of well plates cause the realtive small volumes and high surfaces |
− | elaborate
| + | (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is |
− | a
| + | essential to know the volume in the wells for measuring in the plate reader. Therefore we compared |
− | cultivating and measuring workflow.
| + | different seals for the well plates and in the end we came to the conclusion that using a |
− | <br>
| + | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were |
− | For the cultivating workflow we tested different well plate
| + | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to |
− | formats and growing parameters for the best growing conditions. It was
| + | cultivate the cells for 2-3 days without losing significant amounts of medium. |
− | logistically
| + | <br> |
− | the
| + | <br> |
− | best way to cultivate and measure the parts in well plates, because the Marburg
| + | <center>xxxx |
− | Collection 2.0 comprises xxx parts and we were limited in space in our
| + | Fig x.:Schema vom Workflow</center> |
− | incubator.
| + | <br> |
− | Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
| + | As described before we used the following workflow as shown in fig. XX to cultivate and measure |
− | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the
| + | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the |
− | cultures
| + | end of the triparental conjugation (LINK). For every part we picked 3 different colonies and |
− | showed small clouds of cells formed by inappropriate movement of media in the
| + | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
− | wells.
| + | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to |
− | In
| + | 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 |
− | addition, the rpm of the incubator was limited whereas cultures in flasks had to
| + | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was |
− | be
| + | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while |
− | incubated at the same time and these threatened to fall over at high rpm. At 130
| + | evaluating the results (that will be used as a blank while ...). When all the cultures in the |
− | rpm
| + | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same |
− | we
| + | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating |
− | found a compromise between cultivating flasks and well-plates in the same
| + | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When |
− | incubator.
| + | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
− | After revising the workflow over and over we came to the conclusion, that it is
| + | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three |
− | favorable to cultivate the UTEX 2973 in transparent 24-well-plates because there
| + | times. Following this workflow we were able to measure three biological parallels and |
− | was
| + | two technical parallels for every biological parallel. It enabled us to have a good statistical |
− | enough movement in the wells to prevent the cells from forming a pellet/cloud.
| + | database and gives our results a stronger meaning/significance. While working with this workflow |
− | Further
| + | it was essential to keep the cultures in their exponential phase because it would significantly |
− | it was necessary to use transparent wells to ensure every well with similar
| + | speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es |
− | light
| + | erst gar keine lag phase gibt).<br> |
− | conditions. Concerning of light conditions, we evaluated that the cells showed
| + | Concerning the measurement part we decided to transfer the cultures into black/white luminescence |
− | good
| + | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every |
− | (prosperous?) growth in the wells at low-light conditions (around 500 µE). The
| + | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure |
− | evaporation of medium plays an important role in cultivation of well plates
| + | eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for |
− | cause
| + | measurement)<br> |
− | the
| + | <br> |
− | realtive small volumes and high surfaces (ich glaub die flache ist eher klein,
| + | <b>Fluorescence measurement:</b><br> |
− | aber
| + | After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. |
− | vllt
| + | More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP |
− | wegen der Temperatur und Zeit?). Further it is essential to know the volume in
| + | fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the |
− | the
| + | considered part. For measurement we created a program that measured the OD<sub>730</sub> and the |
− | wells
| + | fluorescence of the wells.<br> |
− | for measuring in the plate reader. Therefore we compared different seals for the
| + | <br> |
− | well
| + | <center>fig XX (screenshot des messprogams)</center> |
− | plates and in the end we came to the conclusion that using a semipermeable foil
| + | <br> |
− | is
| + | In order to measure the OD in each well we determined the absorption at 730 nm. Further we |
− | the
| + | measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the |
− | best solution. The evaporation could be minimalized and the cells were able to
| + | border of the well showed consistent results with small standard deviations (fig. XX). We used the |
− | get
| + | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as |
− | enough CO2 because air transfer was provide/permit. By using a foil it was
| + | signal for our part measurement we have set the emission wavelength to 515 nm and the excitation |
− | possible
| + | wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database |
− | to
| + | verlinken/als quelle?)<br> |
− | cultivate the cells for 2-3 days without losing significant amounts of medium.
| + | <br> |
− | <br>
| + | <b>Fluorescence-Activated Cell Sorting (FACS):</b><br> |
− | <br>
| + | short abstract and link to the FACS-text of the measurement |
− | <center>xxxx
| + | <br> |
− | Fig x.:Schema vom Workflow</center>
| + | <br> |
− | <br>
| + | <b>Luminescence Measurement</b><br> |
− | As described before we used the following workflow as shown in fig. XX to
| + | <br> |
− | cultivate
| + | text |
− | and
| + | </p> |
− | measure our parts. The cultivation started by picking colonies from
| + | </div> |
− | 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
| + | |
− | 96-well-plates. While fluorescence is measured in black well-plates the
| + | |
− | 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
| + | |
− | 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> | | </div> |
− | </div>
| + | <br> |
− | <br>
| + | <div class="wrap-collabsible"> |
− | <div class="wrap-collabsible">
| + | <input id="collapsible4_2" class="toggle" type="checkbox"> |
− | <input id="collapsible4_2"
| + | <label for="collapsible4_2" class="lbl-toggle"> |
− | class="toggle"
| + | <h3 class="title" tyle="text-align: left; text-align-last: left;">Data analysis and evaluation |
− | type="checkbox">
| + | </h3> |
− | <label for="collapsible4_2"
| + | </label> |
− | class="lbl-toggle">
| + | <div class="collapsible-content"> |
− | <h3 class="title"
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
− | style="text-align: left; text-align-last: left;">Data analysis and evaluation
| + | <p> |
− | </h3>
| + | kein plan was man hier schreiben soll zum jetzigen standpunkt... |
− | </label>
| + | For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for |
− | <div class="collapsible-content">
| + | the fluorescence measurement we used UTEX 2973 without a fluorescent protein. |
− | <div class="content-inner"
| + | <br> |
− | style="text-align: left; text-align-last: left;">
| + | Auswertung, Daten und Grafen darstellen? |
− | <p>
| + | </p> |
− | kein plan was man hier schreiben soll zum jetzigen standpunkt...
| + | </div> |
− | 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> |
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− | </div>
| + | <div class="sub" onclick="popup('rbn5')"> |
− | <br>
| + | <div class="sub-header"> |
− | <div class="container">
| + | <h1> |
− | <div class="box" | + | G R O W T H<br> |
− | style="cursor: pointer;"
| + | C U R V E S |
− | onclick="popup('rbn4')">
| + | </h1> |
− | <article class="media"> | + | <hr> |
− | <div class="media-content"> | + | </div> |
− | <div class="content"> | + | <div class="sub-content"> |
− | <h2>Growth Curves</h2>
| + | <div> |
− | <p>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | |
− | </p>
| + | |
− | </div>
| + | |
| </div> | | </div> |
− | </article>
| |
− | </div>
| |
− | </div>
| |
− | <div id="rbn4"
| |
− | class="popup">
| |
− | <div class="popup-container">
| |
− | <div class="popup-header">
| |
− | <h1>Growth Curves</h1>
| |
− | <button type="button"
| |
− | onclick="hide('rbn4')">X</button>
| |
| </div> | | </div> |
− | <div class="popup-content" | + | </div> |
− | style="text-align: justify; text-align-last: justify;"> | + | <div id="rbn5" class="popup"> |
− | <p>
| + | <div class="popup-container"> |
− | Abstract?
| + | <div class="popup-header"> |
− | </p>
| + | <h1 class="title">Growth Curves</h1> |
− | <br>
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− | <br>
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− | style="text-align: left; text-align-last: left;">
| + | </label> |
− | <p>
| + | <div class="collapsible-content"> |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
− | ist.
| + | <p> |
− | </p>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| + | </p> |
| + | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | </div>
| + | <br> |
− | <br>
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− | <div class="wrap-collabsible">
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− | </label>
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
− | <div class="collapsible-content">
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− | <div class="content-inner"
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− | style="text-align: left; text-align-last: left;">
| + | |
| <p> | | <p> |
− | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig | + | One mayor criteria to evaluate a chassis is its potential for an application project. To showcase these potential applications, |
− | ist. | + | 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 |
| + | 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>, 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> | | </p> |
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