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| | {{Marburg}} | | {{Marburg}} |
| | <html> | | <html> |
| − | <style> | + | <div> |
| − | .box-dark { | + | <div class="box-dark"> |
| − | background-color: #3d404d;
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| − | min-height: 30vh;
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| − | box-shadow: 1px 1px 40px black;
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| − | margin-left: -10vw;
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| − | .heading {
| + | <h1 class="heading" style=" |
| − | color: #f5f5f5;
| + | line-height: 1; |
| − | text-align: center; | + | "> |
| − | font-size: 1.75em; | + | |
| − | width: fit-content; | + | G I A N T<br> J A M B O R E E |
| − | margin-top: 25px;
| + | </h1> |
| − | margin-bottom: unset !important; | + | <hr class="line"> |
| − | transform: rotate(-355deg); | + | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo"> |
| − | }
| + | </div> |
| | + | <div style="margin-top: 10vh;"> |
| | + | <section class="section"> |
| | + | <div style="display: flex; flex-direction: row;"> |
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| − | .line {
| + | <p style="margin-bottom: 1em;"> |
| − | border-top: 2px solid #f5f5f5;
| + | 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. |
| − | background-color: #f5f5f5;
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| − | width: 100px;
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| − | margin-top: 25px;
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| − | margin-bottom: unset;
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| − | transform: rotate(-355deg);
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| − | }
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| − | .logo {
| + | </p> |
| − | width: 100px;
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| − | height: 100px;
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| − | position: absolute;
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| − | bottom: -50px;
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| − | transform: rotate(-355deg);
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| − | margin-left: -10px;
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| − | }
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| − | .main {
| + | <figure style="text-align: center; margin-left: 25px; max-width: 42000px"> |
| − | overflow-x: hidden;
| + | <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> |
| | | | |
| − | hr {
| + | </div> |
| − | display: block;
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| − | border-top: 2px solid #3d404d;
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| − | margin: 1em auto;
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| − | width: 50vw;
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| − | }
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| − | .hexagon {
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| − | background: url(https://static.igem.org/mediawiki/2019/8/88/T--Marburg--hexagon.svg);
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| − | background-repeat: no-repeat;
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| − | height: 200px;
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| − | width: 200px;
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| − | justify-content: center;
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| − | }
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| − | .media {
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| − | align-items: center !important;
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| − | min-width: 80vw;
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| − | @media (max-width: 700px) {
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| − | @media (max-width: 810px) { | + | <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> |
| | | | |
| − | .logo,
| + | <br> |
| − | .line,
| + | |
| − | .heading {
| + | |
| − | margin-left: -30px;
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| − | }
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| − | .sub {
| + | <br> |
| − | cursor: pointer;
| + | <p style="margin-bottom: 1em;"> |
| − | display: flex;
| + | <br> Afterwards, most of the time we had to stay with three or more people near our |
| − | justify-content: center;
| + | <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> |
| | | | |
| − | .popup-background {
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| − | width: 100vw;
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| − | height: 100vh;
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| − | background-color: rgba(0, 0, 0, .5);
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| − | z-index: 99;
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| | | | |
| − | </style>
| + | |
| − | <div>
| + | <p style="margin-bottom: 1em;"> |
| − | <div class="box-dark">
| + | 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! |
| − | <h1 class="heading">
| + | |
| − | M E A S U R E M E N T
| + | |
| − | </h1>
| + | |
| − | <hr class="line">
| + | |
| − | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg"
| + | |
| − | class="logo"
| + | |
| − | alt="Syntex Logo">
| + | |
| − | </div>
| + | |
| − | <div style="margin-top: 10vh;">
| + | |
| − | <section class="section">
| + | |
| − | <h1 class="title">Amplifying new standards in measurement</h1>
| + | |
| − | <p style="text-align: justify;">
| + | |
| − | Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
| + | |
| | </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> |
| | + | |
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| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | <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"> |
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| | microbiology and necessary to compare results with other scientists and reproduce their data. | | microbiology and necessary to compare results with other scientists and reproduce their data. |
| | </p> | | </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 |
| | + | 2973 Absorption spectra. |
| | + | </figcaption> |
| | + | </figure> |
| | <p style="text-align: justify; margin-bottom: 1em;"> | | <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 | | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to |
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| | </section> | | </section> |
| | <hr> | | <hr> |
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| − | <article class="media"> | + | <h1> |
| − | <div class="media-left"> | + | L I G H T<br> |
| − | <div class="hexagon"> | + | M E A S U R E M E N T |
| − | Light<br>
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| − | measurement
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| + | Abstract? |
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| + | Unterprojekt1 |
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| − | style="text-align: left; text-align-last: left;">
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| | <p> | | <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. |
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| | <p> | | <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. |
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| − | <div class="media-left"> | + | R E P O R T E R S |
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| − | Reporters
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| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="sub" | + | <div class="sub" onclick="popup('rbn3')"> |
| − | onclick="popup('rbn3')">
| + | <div class="sub-header"> |
| − | <article class="media"> | + | <h1> |
| − | <div class="media-left"> | + | F A C S |
| − | <div class="hexagon"> | + | </h1> |
| − | FACS
| + | <hr> |
| − | </div>
| + | </div> |
| | + | <div class=" sub-content"> |
| | + | |
| | + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| | </div> | | </div> |
| − | <div class=" media- content"> | + | </div> |
| − | | + | </div> |
| − | <h2>Fluorescence-Activated Cell Sorting</h2>
| + | <div id="rbn3" class="popup"> |
| − | <p>
| + | <div class="popup-container"> |
| − | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | <div class=" popup-header"> |
| − | </p>
| + | <h1 class="title">Fluorescence-Activated Cell Sorting (FACS)</h1> |
| − | </div>
| + | <button type="button" onclick="hide('rbn3')">X</button> |
| | </div> | | </div> |
| − | </article>
| + | <div class="popup-content" style="text-align: justify; text-align-last: justify;"> |
| − | </div>
| + | |
| − | <div class="popup-background">
| + | Abstract? |
| − | <div id="rbn3"
| + | </p> |
| − | class="popup">
| + | <br> |
| − | <div class="popup-container"> | + | <br> |
| − | <div class="popup-header">
| + | <div class="wrap-collabsible"> |
| − | <h1>Fluorescence-Activated Cell Sorting (FACS)</h1>
| + | <input id="collapsible3_1" class="toggle" type="checkbox"> |
| − | <button type="button"
| + | <label for="collapsible3_1" class="lbl-toggle"> |
| − | onclick="hide('rbn3')">X</button>
| + | <h3 class="title">Unterprojekt1</h3> |
| − | </div>
| + | </label> |
| − | <div class="popup-content" | + | <div class="collapsible-content"> |
| − | style="text-align: justify; text-align-last: justify;">
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| − | | + | |
| − | Abstract?
| + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| − | </p>
| + | </p> |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <div class="wrap-collabsible"> | + | |
| − | <input id="collapsible3_1"
| + | |
| − | class="toggle"
| + | |
| − | type="checkbox">
| + | |
| − | <label for="collapsible3_1"
| + | |
| − | class="lbl-toggle">
| + | |
| − | <h3 class="title">Unterprojekt1</h3>
| + | |
| − | </label>
| + | |
| − | <div class="collapsible-content"> | + | |
| − | <div class="content-inner" | + | |
| − | style="text-align: left; text-align-last: left;">
| + | |
| − | | + | |
| − | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <br>
| + | </div> |
| − | <div class="wrap-collabsible"> | + | <br> |
| − | <input id="collapsible3_2"
| + | <div class="wrap-collabsible"> |
| − | class="toggle"
| + | <input id="collapsible3_2" class="toggle" ype="checkbox"> |
| − | ype="checkbox">
| + | <label for="collapsible3_2" class="lbl-toggle"> |
| − | <label for="collapsible3_2"
| + | <h3 class="title">Unterprojekt2</h3> |
| − | class="lbl-toggle">
| + | </label> |
| − | <h3 class="title">Unterprojekt2</h3>
| + | <div class="collapsible-content"> |
| − | </label>
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| − | <div class="collapsible-content"> | + | |
| − | <div class="content-inner" | + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| − | style="text-align: left; text-align-last: left;">
| + | </p> |
| − | | + | |
| − | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="sub" | + | <div class="sub" onclick="popup('rbn4')"> |
| − | onclick="popup('rbn4')">
| + | <div class="sub-header"> |
| − | <article class="media"> | + | <h1> |
| − | <div class="media-left"> | + | P A R T<br> |
| − | <div class="hexagon"> | + | M E A S U R E M E N T |
| − | Part<br>
| + | </h1> |
| − | measurement
| + | <hr> |
| − | </div> | + | </div> |
| | + | <div class=" sub-content"> |
| | + | <div> |
| | + | 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. |
| | </div> | | </div> |
| − | <div class=" media-content"> | + | </div> |
| − | | + | </div> |
| − | <p>
| + | <div id="rbn4" class="popup"> |
| − | For our project it was indispensable to establish a measurement workflow that is not only applicable
| + | <div class=" popup-container"> |
| − | to UTEX 2973 and other cyanobacteria but also has a high throughput.
| + | <div class="popup-header"> |
| − | </p>
| + | <h1 class="title">Part Measurement</h1> |
| − | </div> | + | <button type="button" onclick="hide('rbn4')">X</button> |
| | </div> | | </div> |
| − | </article>
| + | <div class="popup-content"> |
| − | </div>
| + | <p> |
| − | <div class="popup-background">
| + | For our project it was indispensable to establish a measurement workflow that is not only applicable |
| − | <div id="rbn4"
| + | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg |
| − | class="popup">
| + | Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement |
| − | <div class="popup-container"> | + | method that suites such a large collection. Therefore we elaborated different workflows - containing |
| − | <div class="popup-header"> | + | different cultivation vessels and parameters - and revised them after evaluating the results. In the end |
| − | <h1>Part Measurement</h1> | + | we were able to establish a workflow specially designed for our methods to cultivate and characterize |
| − | <button type="button"
| + | the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX 2973. |
| − | onclick="hide('rbn4')">X</button>
| + | </p> |
| − | </div>
| + | <div class="wrap-collabsible"> |
| − | <div class="popup-content"
| + | <input id="collapsible4_1" class="toggle" type="checkbox"> |
| − | style="text-align: justify; text-align-last: justify;">
| + | <label for="collapsible4_1" class="lbl-toggle"> |
| − | <p>
| + | <h3 class="title" style="text-align: left; text-align-last: left;"> |
| − | For our project it was indispensable to establish a measurement workflow that is not only applicable
| + | Experimental Procedure |
| − | to UTEX 2973 and other cyanobacteria but also has a high throughput. While we worked on our Marburg
| + | </h3> |
| − | Collection 2.0 with XXX parts we came to the conclusion it is also necessary to develop a measurement
| + | </label> |
| − | method that suites such a large collection. Therefore we elaborated different workflows - containing
| + | <div class="collapsible-content"> |
| − | different cultivation vessels and parameters - and revised them after evaluating the results. In the
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| − | end
| + | |
| − | we were able to establish a workflow specially designed for our methods to cultivate and characterize
| + | The results of our part characterization were obtained by fluorescence and luminescence |
| − | the parts from our Marburg Collection 2.0, that is tailored to <i>Synechococcus elongatus</i> UTEX
| + | measurements (of what?). But before the party could be measured we had to |
| − | 2973.
| + | elaborate a cultivating and measuring workflow.<br> |
| − | </p>
| + | For the cultivating workflow we tested different well plate formats and growing parameters for the |
| − | <div class="wrap-collabsible"> | + | best growing conditions. It was logistically the best way to cultivate and measure the parts in |
| − | <input id="collapsible4_1"
| + | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space |
| − | class="toggle"
| + | in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus |
| − | type="checkbox">
| + | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small |
| − | <label for="collapsible4_1"
| + | clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of |
| − | class="lbl-toggle">
| + | the incubator was limited whereas cultures in flasks had to be incubated at the same time and |
| − | <h3 class="title"
| + | these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating |
| − | style="text-align: left; text-align-last: left;">
| + | flasks and well-plates in the same incubator. After revising the workflow over and over we came to |
| − | Experimental Procedure
| + | the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates |
| − | </h3>
| + | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud. |
| − | </label>
| + | Further it was necessary to use transparent wells to ensure every well with similar ight |
| − | <div class="collapsible-content"> | + | conditions. Concerning of light conditions, we evaluated that the cells showed good (prosperous?) |
| − | <div class="content-inner" | + | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an |
| − | style="text-align: left; text-align-last: left;">
| + | 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 |
| − | The results of our part characterization were obtained by fluorescence and luminescence
| + | essential to know the volume in the wells for measuring in the plate reader. Therefore we compared |
| − | measurements (of what?). But before the party could be measured we had to
| + | different seals for the well plates and in the end we came to the conclusion that using a |
| − | elaborate a cultivating and measuring workflow.<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 formats and growing parameters for
| + | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible to |
| − | the
| + | cultivate the cells for 2-3 days without losing significant amounts of medium. |
| − | best growing conditions. It was logistically the best way to cultivate and measure the parts in
| + | <br> |
| − | well plates, because the Marburg Collection 2.0 comprises xxx parts and we were limited in space
| + | <br> |
| − | in our incubator. Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
| + | <center>xxxx |
| − | elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures showed small
| + | Fig x.:Schema vom Workflow</center> |
| − | clouds of cells formed by inappropriate movement of media in the wells. In addition, the rpm of
| + | <br> |
| − | the incubator was limited whereas cultures in flasks had to be incubated at the same time and
| + | As described before we used the following workflow as shown in fig. XX to cultivate and measure |
| − | these threatened to fall over at high rpm. At 130 rpm we found a compromise between cultivating
| + | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at the |
| − | flasks and well-plates in the same incubator. After revising the workflow over and over we came
| + | end of the triparental conjugation (LINK). For every part we picked 3 different colonies and |
| − | to
| + | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates |
| − | the conclusion, that it is favorable to cultivate the UTEX 2973 in transparent 24-well-plates
| + | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to |
| − | because there was enough movement in the wells to prevent the cells from forming a pellet/cloud.
| + | 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 |
| − | Further it was necessary to use transparent wells to ensure every well with similar ight
| + | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was |
| − | conditions. Concerning of light conditions, we evaluated that the cells showed good
| + | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while |
| − | (prosperous?)
| + | evaluating the results (that will be used as a blank while ...). When all the cultures in the |
| − | growth in the wells at low-light conditions (around 500 µE). The evaporation of medium plays an
| + | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same |
| − | important role in cultivation of well plates cause the realtive small volumes and high surfaces
| + | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating |
| − | (ich glaub die flache ist eher klein, aber vllt wegen der Temperatur und Zeit?). Further it is
| + | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When |
| − | essential to know the volume in the wells for measuring in the plate reader. Therefore we
| + | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred |
| − | compared
| + | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three |
| − | different seals for the well plates and in the end we came to the conclusion that using a
| + | times. Following this workflow we were able to measure three biological parallels and |
| − | semipermeable foil is the best solution. The evaporation could be minimalized and the cells were
| + | two technical parallels for every biological parallel. It enabled us to have a good statistical |
| − | able to get enough CO2 because air transfer was provide/permit. By using a foil it was possible
| + | database and gives our results a stronger meaning/significance. While working with this workflow |
| − | to
| + | it was essential to keep the cultures in their exponential phase because it would significantly |
| − | cultivate the cells for 2-3 days without losing significant amounts of medium.
| + | speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es |
| − | <br>
| + | erst gar keine lag phase gibt).<br> |
| − | <br>
| + | Concerning the measurement part we decided to transfer the cultures into black/white luminescence |
| − | <center>xxxx
| + | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every |
| − | Fig x.:Schema vom Workflow</center>
| + | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could measure |
| − | <br>
| + | eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for |
| − | As described before we used the following workflow as shown in fig. XX to cultivate and measure
| + | measurement)<br> |
| − | our parts. The cultivation started by picking colonies from BG11-agar-plates that were used at
| + | <br> |
| − | the
| + | <b>Fluorescence measurement:</b><br> |
| − | end of the triparental conjugation (LINK). For every part we picked 3 different colonies and
| + | After transfering the cultures into the 96-well-plate the fluorescence of the parts was measured. |
| − | inoculated them in 1.0 mL BG11-media with 0.5 µl Spectinomycin. Thus in the first 24-well-plates
| + | More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP |
| − | we could inoculate 8 different parts with 3 biological parallels. When the cultures grew to
| + | fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as the |
| − | 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
| + | considered part. For measurement we created a program that measured the OD<sub>730</sub> and the |
| − | A1-3 (part 1) and A4-6 (part 2) of another 24-well-plate. At the same time the Well B6 was
| + | fluorescence of the wells.<br> |
| − | inoculated with 1.0 mL of a OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while
| + | <br> |
| − | evaluating the results (that will be used as a blank while ...). When all the cultures in the
| + | <center>fig XX (screenshot des messprogams)</center> |
| − | second 24-well-plate reached OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same
| + | <br> |
| − | well-plate. It was done by inoculating the wells A1-3 into the wells C1-3 and D1-3 creating
| + | In order to measure the OD in each well we determined the absorption at 730 nm. Further we |
| − | technical parallels of the same part (analog for A4-6 and the UDAR inoculating to B4 and B5).
| + | measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the |
| − | When
| + | border of the well showed consistent results with small standard deviations (fig. XX). We used the |
| − | the wells C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred
| + | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as |
| − | into a 96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three
| + | signal for our part measurement we have set the emission wavelength to 515 nm and the excitation |
| − | times. Following this workflow we were able to measure three biological parallels and
| + | wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database |
| − | two technical parallels for every biological parallel. It enabled us to have a good statistical
| + | verlinken/als quelle?)<br> |
| − | database and gives our results a stronger meaning/significance. While working with this workflow
| + | <br> |
| − | it was essential to keep the cultures in their exponential phase because it would significantly
| + | <b>Fluorescence-Activated Cell Sorting (FACS):</b><br> |
| − | speed up the growth by reducing the lag-phase to an absolute minimum (oder lieber sagen dass es
| + | short abstract and link to the FACS-text of the measurement |
| − | erst gar keine lag phase gibt).<br>
| + | <br> |
| − | Concerning the measurement part we decided to transfer the cultures into black/white
| + | <br> |
| − | luminescence
| + | <b>Luminescence Measurement</b><br> |
| − | is measured in white ones. We measured in 96-well-plates because it enabled us to measure every
| + | <br> |
| − | part three times by consuming only 600 µl of the 1.0 ml 24-well-cultures. Further we could
| + | text |
| − | measure
| + | </p> |
| − | eight (?) parts in only one plate. (four 24-well-plates lead into one 96-well-plate for
| + | |
| − | measurement)<br>
| + | |
| − | <br>
| + | |
| − | <b>Fluorescence measurement:</b><br>
| + | |
| − | After transfering the cultures into the 96-well-plate the fluorescence of the parts was
| + | |
| − | measured.
| + | |
| − | More precisely, the activity of the parts was determined by the expression of the sYFP. The sYFP
| + | |
| − | fluorescence served as an indicator and the sequence for the sYFP was in the same cassette as
| + | |
| − | the
| + | |
| − | considered part. For measurement we created a program that measured the OD<sub>730</sub> and the
| + | |
| − | fluorescence of the wells.<br>
| + | |
| − | <br>
| + | |
| − | <center>fig XX (screenshot des messprogams)</center>
| + | |
| − | <br>
| + | |
| − | In order to measure the OD in each well we determined the absorption at 730 nm. Further we
| + | |
| − | measured multiple points in each well, where 3x3 points (circular) with a gap of 1350nm to the
| + | |
| − | border of the well showed consistent results with small standard deviations (fig. XX). We used
| + | |
| − | the
| + | |
| − | same settings of the multiple measurement for the fluorescence measurement. While using sYFP as
| + | |
| − | signal for our part measurement we have set the emission wavelength to 515 nm and the excitation
| + | |
| − | wavelength to 527 nm, fitting the exact wavelengths of the sYFP shown in XX (Database
| + | |
| − | verlinken/als quelle?)<br>
| + | |
| − | <br>
| + | |
| − | <b>Fluorescence-Activated Cell Sorting (FACS):</b><br>
| + | |
| − | short abstract and link to the FACS-text of the measurement
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <b>Luminescence Measurement</b><br>
| + | |
| − | <br>
| + | |
| − | text
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | <br>
| + | </div> |
| − | <div class="wrap-collabsible"> | + | <br> |
| − | <input id="collapsible4_2"
| + | <div class="wrap-collabsible"> |
| − | class="toggle"
| + | <input id="collapsible4_2" class="toggle" type="checkbox"> |
| − | type="checkbox">
| + | <label for="collapsible4_2" class="lbl-toggle"> |
| − | <label for="collapsible4_2"
| + | <h3 class="title" tyle="text-align: left; text-align-last: left;">Data analysis and evaluation |
| − | class="lbl-toggle">
| + | </h3> |
| − | <h3 class="title"
| + | </label> |
| − | tyle="text-align: left; text-align-last: left;">Data analysis and evaluation
| + | <div class="collapsible-content"> |
| − | </h3>
| + | <div class="content-inner" style="text-align: left; text-align-last: left;"> |
| − | </label>
| + | |
| − | <div class="collapsible-content"> | + | kein plan was man hier schreiben soll zum jetzigen standpunkt... |
| − | <div class="content-inner" | + | For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for |
| − | style="text-align: left; text-align-last: left;">
| + | the fluorescence measurement we used UTEX 2973 without a fluorescent protein. |
| − | | + | <br> |
| − | kein plan was man hier schreiben soll zum jetzigen standpunkt...
| + | Auswertung, Daten und Grafen darstellen? |
| − | For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11) and for
| + | </p> |
| − | the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
| + | |
| − | <br>
| + | |
| − | Auswertung, Daten und Grafen darstellen?
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | <div class="sub" | + | <div class="sub" onclick="popup('rbn5')"> |
| − | onclick="popup('rbn4')">
| + | <div class="sub-header"> |
| − | <article class="media"> | + | <h1> |
| − | <div class="media-left"> | + | G R O W T H<br> |
| − | <div class="hexagon"> | + | C U R V E S |
| − | Growth<br>
| + | </h1> |
| − | Curves
| + | <hr> |
| − | </div> | + | </div> |
| | + | <div class=" sub-content"> |
| | + | <div> |
| | + | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist. |
| | </div> | | </div> |
| − | <div class=" media-content"> | + | </div> |
| − | | + | </div> |
| − | <p>
| + | <div id="rbn5" class="popup"> |
| − | Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
| + | <div class=" popup-container"> |
| − | </p>
| + | <div class="popup-header"> |
| − | </div> | + | <h1 class="title">Growth Curves</h1> |
| | + | <button type="button" onclick="hide('rbn5')">X</button> |
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| | + | <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 |
| | + | 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> |
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