Difference between revisions of "Team:Goettingen/Notebook/evolution September"

(Created page with "<div class="notebook-item"> <div class="notebook-head"> <h3 class="notebook-head_title">Growth with glyphosate</h3> <p class="notebook-head_date">28.09.18<...")
 
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         <h3 class="notebook-head_title">Growth with glyphosate</h3>
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         <h3 class="notebook-head_title">Transformation of SP1 with <em>aroA*</em> and <em>gat</em></h3>
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         <p class="notebook-head_date">28.09.18</p>
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         <p class="notebook-head_date">03.09.18</p>
 
     </div>
 
     </div>
 
     <div class="notebook-content">
 
     <div class="notebook-content">
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         <p>Cultures of newly designed strains iGEM37, iGEM38, iGEM39, iGEM40, iGEM41, and SP1 (WT) were prepared in CS-Glc minimal medium. Then, the cultures were transferred in a 96&ndash;well plate with increasing concentrations of glyphosate. The plate layout was as depicted below.</p>
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         <p>The <em>B. subtilis</em> strain SP1 was transformed with correct plasmids that contain either <em>aroA*</em> and <em>gat</em>. After incubation over night at 37 °C transformants were isolated. </p>
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        <div class="article_picture">
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−
            <img src="https://static.igem.org/mediawiki/2018/7/75/T--Goettingen--platereader_280918.png">
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            <p>Strains in 100&nbsp;µL CS-Glc on 96-well plate. The absorption was measured at &lambda;=600&nbsp;nm (OD<sub>600</sub>).</p>
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−
        </div>
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        <p>The results of this experiment is shown <a href="https://2018.igem.org/Team:Goettingen/Results">here</a>!</p>
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     </div>
 
     </div>
 
</div>
 
</div>
 +
<div class="notebook-item">
 +
    <div class="notebook-head">
 +
        <h3 class="notebook-head_title"></h3>
 +
        <p class="notebook-head_date">05.09.18</p>
 +
    </div>
 +
    <div class="notebook-content">
 +
        <p>The positive clones were singularized and afterwards propagated on strach plates in order to check the amylase activity. The starch test was performed as described <a href="https://2018.igem.org/Team:Goettingen/Experiments">here</a>. Positive Strains were isolated for further analysis. </p>
 +
    </div>
 +
</div>
 +
<div class="notebook-item">
 +
    <div class="notebook-head">
 +
        <h3 class="notebook-head_title">Resistence test of <em>aroA*</em> and <em>gat</em> Strains</h3>
 +
        <p class="notebook-head_date">07.09.18</p>
 +
    </div>
 +
    <div class="notebook-content">
 +
        <p>Both constructs, SP1+<em>aroA*</em> as well as <em>gat</em> were propagated on CS-Glucose plates that contain 5 and 10 mM glyphosate. Each plate was incubated for 2 days at 37 °C.</p>
 +
    </div>
 +
    <div class="notebook-table article_table">
 +
        <table>
 +
            <tr>
 +
                <th>Plasmid</th>
 +
                <th class="article-table_right">Resistance</th>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*1</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*2</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*3</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*4</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*5</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*6</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*7</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*8</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*9</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*10</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*11</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*12</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*13</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*14</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*15</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*16</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*17</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*18</em></td>
 +
                <td>&#10008;</td>
 +
            </tr>
 +
            <tr>
 +
                <td><em>aroA*19</em></td>
 +
                <td>&#10004;</td>
 +
            </tr>
 +
            <div class="notebook-content">
 +
                <p>The shown strain variants that have higher resistances than the wildtype strain SP1 are displayed in the table. </p>
 +
            </div>
 +
            <div class="notebook-table article_table">
 +
                <table>
 +
                    <tr>
 +
                        <th>Plasmid</th>
 +
                        <th class="article-table_right">Resistance</th>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>7</td>
 +
                        <td>&#10008;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>9</td>
 +
                        <td>&#10004;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>12</td>
 +
                        <td>&#10004;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>14</td>
 +
                        <td>&#10004;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>18</td>
 +
                        <td>&#10008;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>21</td>
 +
                        <td>&#10008;</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td><em>gat</em>23</td>
 +
                        <td>&#10004;</td>
 +
                    </tr>
 +
                    <div class="notebook-content">
 +
                        <p>The shown strain variants of SP1 with <em>gat</em> that have increased resistance against glyphosate are shown in the following table.</p>
 +
                    </div>
 +
                </table>
 +
            </div>
 +
 +
            <div class="notebook-content">
 +
                <p>The <em>aroA*</em> variants of SP1 are less resistant than the <em>gat</em> variants. The <em>aroA*</em> variants grew on 5 mM glyphosate, whereas the <em>gat</em> variants grew on 10 mM of the herbicide. Two positiv and resistant plasmids of each construct were sequenced additionally.</p>
 +
            </div>
 +
        </table>
 +
    </div>
 +
</div>
 +
<div class="notebook-item">
 +
    <div class="notebook-head">
 +
        <h3 class="notebook-head_title"></h3>
 +
        <p class="notebook-head_date">10.09.18</p>
 +
    </div>
 +
    <div class="notebook-content">
 +
        <p>The variants <em>aroA*</em>10 and <em>gat</em>9 were checked via sequencing and the sequences are correct. Competent cells of the strains SP1+<em>aroA*</em>10 and <em>gat</em>9 were created. Additionally, those strains were cultivated in kryo stocks at -80 °C. </p>
 +
    </div>
 +
</div>
 +
<div class="notebook-item">
 +
    <div class="notebook-head">
 +
        <h3 class="notebook-head_title"></h3>
 +
        <p class="notebook-head_date">11.09.18</p>
 +
    </div>
 +
    <div class="notebook-content">
 +
        <p>The competent strains SP1+<em>aroA*</em>10 and SP1+<em>gat</em>9 were transformed with cDNA of the Strain BP233 (Δ<em>gltT</em>). Additionally, a retransformation of both strains SP1+<em>aroA*</em>10 and SP1+<em>gat</em>9 was performed.</p>
 +
    </div>
 +
</div>
 +
<div class="notebook-item">
 +
    <div class="notebook-head">
 +
        <h3 class="notebook-head_title">Functionality of <em>aroA*</em></h3>
 +
        <p class="notebook-head_date">14.09.18</p>
 +
    </div>
 +
    <div class="notebook-content">
 +
        <p>In order to find out whether the SP1+<em>aroA*</em> veriant that confers glyphosate resistance contains a functional EPSP synthase which is encoded by the <em>aroA*</em> gene. Therefor, the strains SP1 (WT), SP1+<em>aroA*</em> and SP1+<em>aroA*</em> Δ<em>aroE</em> were propagated on CS-Glucose plates.</p>
 +
    </div>
 +
    <div class="article_picture">
 +
        <img src="">
 +
        <p></p>
 +
    </div>

Revision as of 07:55, 4 October 2018

Transformation of SP1 with aroA* and gat

03.09.18

The B. subtilis strain SP1 was transformed with correct plasmids that contain either aroA* and gat. After incubation over night at 37 °C transformants were isolated.

05.09.18

The positive clones were singularized and afterwards propagated on strach plates in order to check the amylase activity. The starch test was performed as described <a href="https://2018.igem.org/Team:Goettingen/Experiments">here</a>. Positive Strains were isolated for further analysis.

Resistence test of aroA* and gat Strains

07.09.18

Both constructs, SP1+aroA* as well as gat were propagated on CS-Glucose plates that contain 5 and 10 mM glyphosate. Each plate was incubated for 2 days at 37 °C.

The shown strain variants that have higher resistances than the wildtype strain SP1 are displayed in the table.

Plasmid Resistance
aroA*1 ✔
aroA*2 ✘
aroA*3 ✘
aroA*4 ✔
aroA*5 ✔
aroA*6 ✔
aroA*7 ✔
aroA*8 ✘
aroA*9 ✘
aroA*10 ✘
aroA*11 ✘
aroA*12 ✘
aroA*13 ✘
aroA*14 ✔
aroA*15 ✔
aroA*16 ✘
aroA*17 ✘
aroA*18 ✘
aroA*19 ✔

The shown strain variants of SP1 with gat that have increased resistance against glyphosate are shown in the following table.

Plasmid Resistance
gat7 ✘
gat9 ✔
gat12 ✔
gat14 ✔
gat18 ✘
gat21 ✘
gat23 ✔

The aroA* variants of SP1 are less resistant than the gat variants. The aroA* variants grew on 5 mM glyphosate, whereas the gat variants grew on 10 mM of the herbicide. Two positiv and resistant plasmids of each construct were sequenced additionally.

10.09.18

The variants aroA*10 and gat9 were checked via sequencing and the sequences are correct. Competent cells of the strains SP1+aroA*10 and gat9 were created. Additionally, those strains were cultivated in kryo stocks at -80 °C.

11.09.18

The competent strains SP1+aroA*10 and SP1+gat9 were transformed with cDNA of the Strain BP233 (ΔgltT). Additionally, a retransformation of both strains SP1+aroA*10 and SP1+gat9 was performed.

Functionality of aroA*

14.09.18

In order to find out whether the SP1+aroA* veriant that confers glyphosate resistance contains a functional EPSP synthase which is encoded by the aroA* gene. Therefor, the strains SP1 (WT), SP1+aroA* and SP1+aroA* ΔaroE were propagated on CS-Glucose plates.

       <img src="">