Difference between revisions of "Team:Marburg/test"

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{{Marburg}}
 
{{Marburg}}
 
<html>
 
<html>
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<body style="overflow: auto; max-height: 100vh;">
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    <style>
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    <center>
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        .box-dark {
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         <h1 class="title">Measurement</h1>
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            background-color: #3d404d;
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    </center>
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            min-height: 60vh;
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    <br>
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            box-shadow: 1px 1px 40px black, 1px 1px 40px black;
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    <br>
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            margin-left: -10vw;
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    <p>
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            width: 120vw;
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         <h1 class="subtitle">Amplifying new standards in measurement</h1><br>
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            position: relative;
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         <p style="text-align: justify;">
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            z-index: 2;
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             <br>
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            display: flex;
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             Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
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            flex-direction: column;
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             <br>
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            align-items: center;
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            <br>
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         }
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         </p>
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−
        <h1 class="subtitle">Storytelling:</h1><br>
+
        .heading {
 +
            color: #f5f5f5;
 +
            text-align: center;
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            font-size: 1.75em;
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            width: fit-content;
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            margin-top: 25px;
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            margin-bottom: unset !important;
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         }
 +
 
 +
         .line {
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            background-color: #f5f5f5;
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             border-width: 2px;
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             display: block;
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             width: 100px;
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            margin-top: 25px;
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            margin-bottom: unset;
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        }
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    </style>
 +
    <div>
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         <div class="box-dark">
 +
            <h1 class="heading">
 +
                M E A S U R E M E N T
 +
            </h1>
 +
            <hr class="line">
 +
        </div>
 
         <br>
 
         <br>
−
         <p style="text-align: justify;">
+
         <br>
−
            We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 2973, the
+
        <p>
−
            fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in front
+
            <h1 class="subtitle">Amplifying new standards in measurement</h1><br>
−
            of
+
            <p style="text-align: justify;">
−
            many problems and questions. Especially the usage of different media, light conditions and other cultivating
+
                <br>
−
             and
+
                Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
−
             measurement parameters were one of the biggest problems we discovered in scientific papers. Many of these
+
                <br>
−
            problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore it
+
                <br>
−
            is
+
             </p>
−
            hard to hold on to special methods but still standardization is a huge part in synthetic microbiology and
+
             <h1 class="subtitle">Storytelling:</h1><br>
−
            necessary to compare results with other scientists and reproduce their data.
+
 
             <br>
 
             <br>
−
             <br>
+
             <p style="text-align: justify;">
−
            While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to
+
                We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 2973,
−
            show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore
+
                the
−
            we
+
                fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in
−
            analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
+
                front
−
            light
+
                of
−
            measurement, evaluated different reporters???, established a measurement method and compared it to a already
+
                many problems and questions. Especially the usage of different media, light conditions and other
−
            known FACS measurement method (?).
+
                cultivating
−
            <br>
+
                and
−
            <br>
+
                measurement parameters were one of the biggest problems we discovered in scientific papers. Many of
−
            At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle].
+
                these
−
            So we
+
                problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore
−
            had to measure the light conditions in our incubators and while doing this simple task the first part of
+
                it
−
            standardization began. We discovered that nearly every paper? is using different methods to measure their
+
                is
−
            light
+
                hard to hold on to special methods but still standardization is a huge part in synthetic microbiology
−
            conditions and that it is a really complex and important procedure. So we got in contact with cyano and
+
                and
−
            light
+
                necessary to compare results with other scientists and reproduce their data.
−
            measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show
+
                <br>
−
            different ways of measurement, their (dis-)advantages and different results depending on the measuring
+
                <br>
−
            instrument.<br>
+
                While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we
−
            Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In
+
                wanted to
−
            literature and while talking with different experts (IHP), we recognized that small deviations of these
+
                show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973.
−
            parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as
+
                Therefore
−
            a
+
                we
−
            new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters
+
                analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
−
            that
+
                light
−
            lead to the fastest growth speed.<br>
+
                measurement, evaluated different reporters???, established a measurement method and compared it to a
−
            Another aspect was measuring the expression and characterize our part. Different possibilities were
+
                already
−
            discussed
+
                known FACS measurement method (?).
−
            and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to
+
−
            measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to
+
−
            standard equipment of every lab nowadays, and could deliver easy reproducible results.<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.
+
−
            <br>
+
−
        </p>
+
−
    </p>
+
−
    <br>
+
−
    <div class="container">
+
−
        <div class="box" style="cursor: pointer;" onclick="popup('rbn1')">
+
−
            <article class="media">
+
−
                <div class="media-content">
+
−
                    <div class="content" style="text-align: left; text-align-last: left;">
+
−
                        <h2>Light measurement</h2>
+
−
                        <p>
+
−
                            Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
+
−
                        </p>
+
−
                    </div>
+
−
                </div>
+
−
            </article>
+
−
        </div>
+
−
    </div>
+
−
    <div id="rbn1" class="popup">
+
−
        <div class="popup-container">
+
−
            <div class="popup-header">
+
−
                <h1>Light Measurement</h1>
+
−
                <button type="button" onclick="hide('rbn1')">X</button>
+
−
            </div>
+
−
            <div class="popup-content" style="text-align: justify; text-align-last: justify;">
+
−
                <p>
+
−
                    Abstract?
+
−
                </p>
+
 
                 <br>
 
                 <br>
 
                 <br>
 
                 <br>
−
                 <div class="wrap-collabsible">
+
                 At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE.
−
                    <input id="collapsible1_1" class="toggle" type="checkbox">
+
                [quelle].
−
                    <label for="collapsible1_1" class="lbl-toggle">Unterprojekt1</label>
+
                So we
−
                     <div class="collapsible-content">
+
                had to measure the light conditions in our incubators and while doing this simple task the first part of
−
                         <div class="content-inner" style="text-align: left; text-align-last: left;">
+
                standardization began. We discovered that nearly every paper? is using different methods to measure
 +
                their
 +
                light
 +
                conditions and that it is a really complex and important procedure. So we got in contact with cyano and
 +
                light
 +
                measurement experts [link IHP] to confront this problem and standardize it. In the following popup we
 +
                show
 +
                different ways of measurement, their (dis-)advantages and different results depending on the measuring
 +
                instrument.<br>
 +
                Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed.
 +
                In
 +
                literature and while talking with different experts (IHP), we recognized that small deviations of these
 +
                parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX
 +
                2973 as
 +
                a
 +
                new chassis we evaluated this impact on the growth speed and were able to show combinations of
 +
                parameters
 +
                that
 +
                lead to the fastest growth speed.<br>
 +
                Another aspect was measuring the expression and characterize our part. Different possibilities were
 +
                discussed
 +
                and after testing them we decided on two methods in our project (plate reader and FACs). One approach
 +
                was to
 +
                measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong
 +
                to
 +
                standard equipment of every lab nowadays, and could deliver easy reproducible results.<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.
 +
                <br>
 +
            </p>
 +
        </p>
 +
        <br>
 +
        <div class="container">
 +
            <div class="box"
 +
                style="cursor: pointer;"
 +
                onclick="popup('rbn1')">
 +
                <article class="media">
 +
                     <div class="media-content">
 +
                         <div class="content"
 +
                            style="text-align: left; text-align-last: left;">
 +
                            <h2>Light measurement</h2>
 
                             <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>
 
                         </div>
 
                     </div>
 
                     </div>
 +
                </article>
 +
            </div>
 +
        </div>
 +
        <div id="rbn1"
 +
            class="popup">
 +
            <div class="popup-container">
 +
                <div class="popup-header">
 +
                    <h1>Light Measurement</h1>
 +
                    <button type="button"
 +
                        onclick="hide('rbn1')">X</button>
 
                 </div>
 
                 </div>
−
                 <br>
+
                 <div class="popup-content"
−
                <div class="wrap-collabsible">
+
                    style="text-align: justify; text-align-last: justify;">
−
                     <input id="collapsible1_2" class="toggle" type="checkbox">
+
                    <p>
−
                    <label for="collapsible1_2" class="lbl-toggle">
+
                        Abstract?
−
                        <h3 class="title">Unterprojekt2</h3>
+
                    </p>
−
                    </label>
+
                    <br>
−
                    <div class="collapsible-content">
+
                    <br>
−
                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+
                    <div class="wrap-collabsible">
−
                            <p>
+
                        <input id="collapsible1_1"
−
                                Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
+
                            class="toggle"
−
                            </p>
+
                            type="checkbox">
 +
                        <label for="collapsible1_1"
 +
                            class="lbl-toggle">Unterprojekt1</label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 +
                        </div>
 +
                     </div>
 +
                    <br>
 +
                    <div class="wrap-collabsible">
 +
                        <input id="collapsible1_2"
 +
                            class="toggle"
 +
                            type="checkbox">
 +
                        <label for="collapsible1_2"
 +
                            class="lbl-toggle">
 +
                            <h3 class="title">Unterprojekt2</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
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             </div>
 
             </div>
 
         </div>
 
         </div>
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    </div>
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        <br>
−
    <br>
+
        <div class="container">
−
    <div class="container">
+
            <div class="box"
−
        <div class="box" style="cursor: pointer;" onclick="popup('rbn2')">
+
                style="cursor: pointer;"
−
            <article class="media">
+
                onclick="popup('rbn2')">
−
                <div class="media-content">
+
                <article class="media">
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                    <div class="content">
+
                    <div class="media-content">
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                        <h2>Reporters</h2>
+
                        <div class="content">
−
                        <p>
+
                            <h2>Reporters</h2>
−
                            Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
+
−
                        </p>
+
−
                    </div>
+
−
                </div>
+
−
            </article>
+
−
        </div>
+
−
    </div>
+
−
    <div id="rbn2" class="popup">
+
−
        <div class="popup-container">
+
−
            <div class="popup-header">
+
−
                <h1>Reporter</h1>
+
−
                <button type="button" onclick="hide('rbn2')">X</button>
+
−
            </div>
+
−
            <div class="popup-content" style="text-align: justify; text-align-last: justify;">
+
−
                <p>
+
−
                    Abstract?
+
−
                </p>
+
−
                <br>
+
−
                <br>
+
−
                <div class="wrap-collabsible">
+
−
                    <input id="collapsible2_1" class="toggle" type="checkbox">
+
−
                    <label for="collapsible2_1" class="lbl-toggle">
+
−
                        <h3 class="title">Unterprojekt1</h3>
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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>
 
                     </div>
 
                     </div>
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        </div>
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        <div id="rbn2"
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                    <h1>Reporter</h1>
 +
                    <button type="button"
 +
                        onclick="hide('rbn2')">X</button>
 
                 </div>
 
                 </div>
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                 <br>
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                 <div class="popup-content"
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                    style="text-align: justify; text-align-last: justify;">
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                     <input id="collapsible2_2" class="toggle" type="checkbox">
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                    <p>
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                    <label for="collapsible2_2" class="lbl-toggle">
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                        Abstract?
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                        <h3 class="title">Unterprojekt2</h3>
+
                    </p>
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                    </label>
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                            <p>
+
                        <input id="collapsible2_1"
−
                                Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
+
                            class="toggle"
−
                            </p>
+
                            type="checkbox">
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                        <label for="collapsible2_1"
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                            class="lbl-toggle">
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                            <h3 class="title">Unterprojekt1</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 +
                        </div>
 +
                     </div>
 +
                    <br>
 +
                    <div class="wrap-collabsible">
 +
                        <input id="collapsible2_2"
 +
                            class="toggle"
 +
                            type="checkbox">
 +
                        <label for="collapsible2_2"
 +
                            class="lbl-toggle">
 +
                            <h3 class="title">Unterprojekt2</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 
                         </div>
 
                         </div>
 
                     </div>
 
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                style="cursor: pointer;"
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            <article class="media">
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                onclick="popup('rbn3')">
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                <div class="media-content">
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                <article class="media">
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                    <div class="content">
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                        <h2>Fluorescence-Activated Cell Sorting (FACS)</h2>
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                        <div class="content">
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                        <p>
+
                            <h2>Fluorescence-Activated Cell Sorting (FACS)</h2>
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                            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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                <h1>Fluorescence-Activated Cell Sorting (FACS)</h1>
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                <button type="button" onclick="hide('rbn3')">X</button>
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            </div>
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                <p>
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                    Abstract?
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                </p>
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                <br>
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                <br>
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                    <input id="collapsible3_1" class="toggle" type="checkbox">
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                        <h3 class="title">Unterprojekt1</h3>
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                    <div class="collapsible-content">
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                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+
 
                             <p>
 
                             <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>
 
                         </div>
 
                     </div>
 
                     </div>
 +
                </article>
 +
            </div>
 +
        </div>
 +
        <div id="rbn3"
 +
            class="popup">
 +
            <div class="popup-container">
 +
                <div class="popup-header">
 +
                    <h1>Fluorescence-Activated Cell Sorting (FACS)</h1>
 +
                    <button type="button"
 +
                        onclick="hide('rbn3')">X</button>
 
                 </div>
 
                 </div>
−
                 <br>
+
                 <div class="popup-content"
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                <div class="wrap-collabsible">
+
                    style="text-align: justify; text-align-last: justify;">
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                     <input id="collapsible3_2" class="toggle" type="checkbox">
+
                    <p>
−
                    <label for="collapsible3_2" class="lbl-toggle">
+
                        Abstract?
−
                        <h3 class="title">Unterprojekt2</h3>
+
                    </p>
−
                    </label>
+
                    <br>
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                    <div class="collapsible-content">
+
                    <br>
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                        <div class="content-inner" style="text-align: left; text-align-last: left;">
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                    <div class="wrap-collabsible">
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                            <p>
+
                        <input id="collapsible3_1"
−
                                Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
+
                            class="toggle"
−
                            </p>
+
                            type="checkbox">
 +
                        <label for="collapsible3_1"
 +
                            class="lbl-toggle">
 +
                            <h3 class="title">Unterprojekt1</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 +
                        </div>
 +
                     </div>
 +
                    <br>
 +
                    <div class="wrap-collabsible">
 +
                        <input id="collapsible3_2"
 +
                            class="toggle"
 +
                            type="checkbox">
 +
                        <label for="collapsible3_2"
 +
                            class="lbl-toggle">
 +
                            <h3 class="title">Unterprojekt2</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    Hier bitte den für diese Stelle zutreffenden Text einfügen, wenn dieser fertig ist.
 +
                                </p>
 +
                            </div>
 
                         </div>
 
                         </div>
 
                     </div>
 
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         </div>
 
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        <br>
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    <br>
+
        <div class="container">
−
    <div class="container">
+
            <div class="box"
−
        <div class="box" style="cursor: pointer;" onclick="popup('rbn4')">
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                style="cursor: pointer;"
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            <article class="media">
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                onclick="popup('rbn4')">
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                <div class="media-content">
+
                <article class="media">
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                    <div class="content">
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                    <div class="media-content">
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                        <h2>Part Measurement</h2>
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                        <div class="content">
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                        <p>
+
                            <h2>Part Measurement</h2>
−
                            For our project it was indispensable to establish a measurement workflow that is not only
+
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                            applicable to UTEX 2973 and other cyanobacteria but also has a high throughput.
+
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                        </p>
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                    </div>
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    </div>
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    <div id="rbn4" class="popup">
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        <div class="popup-container">
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            <div class="popup-header">
+
−
                <h1>Part Measurement</h1>
+
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                <button type="button" onclick="hide('rbn4')">X</button>
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            </div>
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            <div class="popup-content" style="text-align: justify; text-align-last: justify;">
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                <p>
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                    For our project it was indispensable to establish a measurement workflow that is not only applicable
+
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                    to UTEX 2973 and other cyanobacteria but also has a high throughput.
+
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                    While we worked on our Marburg Collection 2.0 with XXX parts we came to the conclusion it is also
+
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                    necessary to develop a measurement method that suites such a large collection. Therefore we
+
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                    elaborated different workflows - containing different cultivation vessels and parameters - and
+
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                    revised them after evaluating the results. In the end we were able to establish a workflow specially
+
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                    designed for our methods to cultivate and characterize the parts from our Marburg Collection 2.0,
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                    that is tailored to <i>Synechococcus elongatus</i> UTEX 2973.
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                </p>
+
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                <br>
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                        <h3 class="title" style="text-align: left; text-align-last: left;">Experimental Procedure</h3>
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                    </label>
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                    <div class="collapsible-content">
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                        <div class="content-inner" style="text-align: left; text-align-last: left;">
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                             <p>
 
                             <p>
−
                                The results of our part characterization were obtained by fluorescence and luminescence
+
                                 For our project it was indispensable to establish a measurement workflow that is not
−
                                measurements (of what?). But before the party could be measured we had to elaborate a
+
                                 only
−
                                cultivating and measuring workflow.
+
                                applicable to UTEX 2973 and other cyanobacteria but also has a high throughput.
−
                                <br>
+
−
                                 For the cultivating workflow we tested different well plate
+
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                                formats and growing parameters for the best growing conditions. It was logistically the
+
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                                best way to cultivate and measure the parts in well plates, because the Marburg
+
−
                                Collection 2.0 comprises xxx parts and we were limited in space in our incubator.
+
−
                                Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
+
−
                                    elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the cultures
+
−
                                showed small clouds of cells formed by inappropriate movement of media in the wells. In
+
−
                                addition, the rpm of the incubator was limited whereas cultures in flasks had to be
+
−
                                incubated at the same time and these threatened to fall over at high rpm. At 130 rpm we
+
−
                                found a compromise between cultivating flasks and well-plates in the same incubator.
+
−
                                After revising the workflow over and over we came to the conclusion, that it is
+
−
                                 favorable to cultivate the UTEX 2973 in transparent 24-well-plates because there was
+
−
                                enough movement in the wells to prevent the cells from forming a pellet/cloud. Further
+
−
                                it was necessary to use transparent wells to ensure every well with similar light
+
−
                                conditions. Concerning of light conditions, we evaluated that the cells showed good
+
−
                                (prosperous?) growth in the wells at low-light conditions (around 500 µE). The
+
−
                                evaporation of medium plays an important role in cultivation of well plates cause the
+
−
                                realtive small volumes and high surfaces (ich glaub die flache ist eher klein, aber vllt
+
−
                                wegen der Temperatur und Zeit?). Further it is essential to know the volume in the wells
+
−
                                for measuring in the plate reader. Therefore we compared different seals for the well
+
−
                                plates and in the end we came to the conclusion that using a semipermeable foil is the
+
−
                                best solution. The evaporation could be minimalized and the cells were able to get
+
−
                                enough CO2 because air transfer was provide/permit. By using a foil it was possible to
+
−
                                cultivate the cells for 2-3 days without losing significant amounts of medium.
+
−
                                <br>
+
−
                                <br>
+
−
                                <center>xxxx
+
−
                                    Fig x.:Schema vom Workflow</center>
+
−
                                <br>
+
−
                                As described before we used the following workflow as shown in fig. XX to cultivate and
+
−
                                measure our parts. The cultivation started by picking colonies from BG11-agar-plates
+
−
                                that were used at the end of the triparental conjugation (LINK). For every part we
+
−
                                picked 3 different colonies and inoculated them in 1.0 mL BG11-media with 0.5 µl
+
−
                                Spectinomycin. Thus in the first 24-well-plates we could inoculate 8 different parts
+
−
                                with 3 biological parallels. When the cultures grew to OD<sub>730</sub>=0.6-0.8 they
+
−
                                were
+
−
                                inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells A1-3 (part 1) and A4-6 (part
+
−
                                2) of
+
−
                                another 24-well-plate. At the same time the Well B6 was inoculated with 1.0 mL of a
+
−
                                OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while evaluating the results
+
−
                                (that will
+
−
                                be used as a blank while ...). When all the cultures in the second 24-well-plate reached
+
−
                                OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same well-plate. It was done
+
−
                                by
+
−
                                inoculating the wells A1-3 into the wells C1-3 and D1-3 creating technical parallels of
+
−
                                the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When the wells
+
−
                                C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were transferred
+
−
                                into a
+
−
                                96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured three
+
−
                                times. Following this workflow we were able to measure three biological parallels and
+
−
                                two technical parallels for every biological parallel. It enabled us to have a good
+
−
                                statistical database and gives our results a stronger meaning/significance. While
+
−
                                working with this workflow it was essential to keep the cultures in their exponential
+
−
                                phase because it would significantly speed up the growth by reducing the lag-phase to an
+
−
                                absolute minimum (oder lieber sagen dass es erst gar keine lag phase gibt).
+
−
                                <br>
+
−
                                Concerning the measurement part we decided to transfer the cultures into black/white
+
−
                                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>
 
                             </p>
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
 +
                </article>
 +
            </div>
 +
        </div>
 +
        <div id="rbn4"
 +
            class="popup">
 +
            <div class="popup-container">
 +
                <div class="popup-header">
 +
                    <h1>Part Measurement</h1>
 +
                    <button type="button"
 +
                        onclick="hide('rbn4')">X</button>
 
                 </div>
 
                 </div>
−
                 <br>
+
                 <div class="popup-content"
−
                <div class="wrap-collabsible">
+
                    style="text-align: justify; text-align-last: justify;">
−
                     <input id="collapsible4_2" class="toggle" type="checkbox">
+
                    <p>
−
                    <label for="collapsible4_2" class="lbl-toggle">
+
                        For our project it was indispensable to establish a measurement workflow that is not only
−
                        <h3 class="title" style="text-align: left; text-align-last: left;">Data analysis and evaluation
+
                        applicable
−
                        </h3>
+
                        to UTEX 2973 and other cyanobacteria but also has a high throughput.
−
                    </label>
+
                        While we worked on our Marburg Collection 2.0 with XXX parts we came to the conclusion it is
−
                    <div class="collapsible-content">
+
                        also
−
                        <div class="content-inner" style="text-align: left; text-align-last: left;">
+
                        necessary to develop a measurement method that suites such a large collection. Therefore we
−
                            <p>
+
                        elaborated different workflows - containing different cultivation vessels and parameters - and
−
                                kein plan was man hier schreiben soll zum jetzigen standpunkt...
+
                        revised them after evaluating the results. In the end we were able to establish a workflow
−
                                For analyzing the data we used two blanks. For OD measurement we used pure medium (BG11)
+
                        specially
−
                                and for the fluorescence measurement we used UTEX 2973 without a fluorescent protein.
+
                        designed for our methods to cultivate and characterize the parts from our Marburg Collection
−
                                <br>
+
                        2.0,
−
                                Auswertung, Daten und Grafen darstellen?
+
                        that is tailored to <i>Synechococcus elongatus</i> UTEX 2973.
−
                            </p>
+
                    </p>
 +
                    <br>
 +
                    <br>
 +
                    <div class="wrap-collabsible">
 +
                        <input id="collapsible4_1"
 +
                            class="toggle"
 +
                            type="checkbox">
 +
                        <label for="collapsible4_1"
 +
                            class="lbl-toggle">
 +
                            <h3 class="title"
 +
                                style="text-align: left; text-align-last: left;">Experimental Procedure</h3>
 +
                        </label>
 +
                        <div class="collapsible-content">
 +
                            <div class="content-inner"
 +
                                style="text-align: left; text-align-last: left;">
 +
                                <p>
 +
                                    The results of our part characterization were obtained by fluorescence and
 +
                                    luminescence
 +
                                    measurements (of what?). But before the party could be measured we had to elaborate
 +
                                    a
 +
                                    cultivating and measuring workflow.
 +
                                    <br>
 +
                                    For the cultivating workflow we tested different well plate
 +
                                    formats and growing parameters for the best growing conditions. It was logistically
 +
                                    the
 +
                                    best way to cultivate and measure the parts in well plates, because the Marburg
 +
                                    Collection 2.0 comprises xxx parts and we were limited in space in our incubator.
 +
                                    Starting with 96-well-plates it was impossible to cultivate <i>Synechococcus
 +
                                        elongatus</i> UTEX 2973 under our conditions (hier aufführen?) since the
 +
                                    cultures
 +
                                    showed small clouds of cells formed by inappropriate movement of media in the wells.
 +
                                    In
 +
                                    addition, the rpm of the incubator was limited whereas cultures in flasks had to be
 +
                                    incubated at the same time and these threatened to fall over at high rpm. At 130 rpm
 +
                                    we
 +
                                    found a compromise between cultivating flasks and well-plates in the same incubator.
 +
                                    After revising the workflow over and over we came to the conclusion, that it is
 +
                                    favorable to cultivate the UTEX 2973 in transparent 24-well-plates because there was
 +
                                    enough movement in the wells to prevent the cells from forming a pellet/cloud.
 +
                                    Further
 +
                                    it was necessary to use transparent wells to ensure every well with similar light
 +
                                    conditions. Concerning of light conditions, we evaluated that the cells showed good
 +
                                    (prosperous?) growth in the wells at low-light conditions (around 500 µE). The
 +
                                    evaporation of medium plays an important role in cultivation of well plates cause
 +
                                    the
 +
                                    realtive small volumes and high surfaces (ich glaub die flache ist eher klein, aber
 +
                                    vllt
 +
                                    wegen der Temperatur und Zeit?). Further it is essential to know the volume in the
 +
                                    wells
 +
                                    for measuring in the plate reader. Therefore we compared different seals for the
 +
                                    well
 +
                                    plates and in the end we came to the conclusion that using a semipermeable foil is
 +
                                    the
 +
                                    best solution. The evaporation could be minimalized and the cells were able to get
 +
                                    enough CO2 because air transfer was provide/permit. By using a foil it was possible
 +
                                    to
 +
                                    cultivate the cells for 2-3 days without losing significant amounts of medium.
 +
                                    <br>
 +
                                    <br>
 +
                                    <center>xxxx
 +
                                        Fig x.:Schema vom Workflow</center>
 +
                                    <br>
 +
                                    As described before we used the following workflow as shown in fig. XX to cultivate
 +
                                    and
 +
                                    measure our parts. The cultivation started by picking colonies from BG11-agar-plates
 +
                                    that were used at the end of the triparental conjugation (LINK). For every part we
 +
                                    picked 3 different colonies and inoculated them in 1.0 mL BG11-media with 0.5 µl
 +
                                    Spectinomycin. Thus in the first 24-well-plates we could inoculate 8 different parts
 +
                                    with 3 biological parallels. When the cultures grew to OD<sub>730</sub>=0.6-0.8 they
 +
                                    were
 +
                                    inoculated to 1.0 mL of OD<sub>730</sub>=0.1 into the wells A1-3 (part 1) and A4-6
 +
                                    (part
 +
                                    2) of
 +
                                    another 24-well-plate. At the same time the Well B6 was inoculated with 1.0 mL of a
 +
                                    OD<sub>730</sub>= 0.1 UDAR culture that was used as a blank while evaluating the
 +
                                    results
 +
                                    (that will
 +
                                    be used as a blank while ...). When all the cultures in the second 24-well-plate
 +
                                    reached
 +
                                    OD<sub>730</sub>=0.6-0.8 they got inoculated twice in the same well-plate. It was
 +
                                    done
 +
                                    by
 +
                                    inoculating the wells A1-3 into the wells C1-3 and D1-3 creating technical parallels
 +
                                    of
 +
                                    the same part (analog for A4-6 and the UDAR inoculating to B4 and B5). When the
 +
                                    wells
 +
                                    C1-D6 (and the UDAR) reached an OD<sub>730</sub>=0.6-0.8 the cultures were
 +
                                    transferred
 +
                                    into a
 +
                                    96-well-plate. As seen in fig. XXX every well of the 24-well-plate was measured
 +
                                    three
 +
                                    times. Following this workflow we were able to measure three biological parallels
 +
                                    and
 +
                                    two technical parallels for every biological parallel. It enabled us to have a good
 +
                                    statistical database and gives our results a stronger meaning/significance. While
 +
                                    working with this workflow it was essential to keep the cultures in their
 +
                                    exponential
 +
                                    phase because it would significantly speed up the growth by reducing the lag-phase
 +
                                    to an
 +
                                    absolute minimum (oder lieber sagen dass es erst gar keine lag phase gibt).
 +
                                    <br>
 +
                                    Concerning the measurement part we decided to transfer the cultures into black/white
 +
                                    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>
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                                    kein plan was man hier schreiben soll zum jetzigen standpunkt...
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                                    For analyzing the data we used two blanks. For OD measurement we used pure medium
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                                    (BG11)
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                                    and for the fluorescence measurement we used UTEX 2973 without a fluorescent
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                                    protein.
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                                    Auswertung, Daten und Grafen darstellen?
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Revision as of 13:39, 19 October 2019

M E A S U R E M E N T



Amplifying new standards in measurement



Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)

Storytelling:



We entered this project as the first Marburg iGEM team working with Synechococcus elongatus UTEX 2973, the fastest phototrophic organism. Missing knowledge in handling and cultivation of UTEX 2973 left us in front of many problems and questions. Especially the usage of different media, light conditions and other cultivating and measurement parameters were one of the biggest problems we discovered in scientific papers. Many of these problems are reasoned in the ongoing optimization and development of methods and instruments. Therefore it is hard to hold on to special methods but still standardization is a huge part in synthetic microbiology and necessary to compare results with other scientists and reproduce their data.

While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in light measurement, evaluated different reporters???, established a measurement method and compared it to a already known FACS measurement method (?).

At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle]. So we had to measure the light conditions in our incubators and while doing this simple task the first part of standardization began. We discovered that nearly every paper? is using different methods to measure their light conditions and that it is a really complex and important procedure. So we got in contact with cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following popup we show different ways of measurement, their (dis-)advantages and different results depending on the measuring instrument.
Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed. In literature and while talking with different experts (IHP), we recognized that small deviations of these parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters that lead to the fastest growth speed.
Another aspect was measuring the expression and characterize our part. Different possibilities were discussed and after testing them we decided on two methods in our project (plate reader and FACs). One approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.
The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the difference in measurement methods.
At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973 and make a contribution to the cyano community by establishing essential/fixed standards in measurement.


Light measurement

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


Reporters

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


Fluorescence-Activated Cell Sorting (FACS)

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


Part Measurement

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.


Growth Curves

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