Difference between revisions of "Team:HZAU-China/Experiments"

 
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                 <div class="h1">Material</div>
 
                 <div class="h1">Material</div>
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−
                 <div class="h2"><b>1.</b>Bacteria Strains used in this work</div>
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                 <div class="h2"><b>1. </b>Bacteria Strains used in this work</div>
 
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                             <th>Description</th>
 
                             <th>Description</th>
 
                             <th>Usage</th>
 
                             <th>Usage</th>
−
<th>Sourse</th>
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                            <th>Sourse</th>
 
                         </tr>
 
                         </tr>
 
                     </thead>
 
                     </thead>
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                             <td><i>E.coli</i> trans-5α</td>
 
                             <td><i>E.coli</i> trans-5α</td>
 
                             <td class="success">Basic strain of calcium conversion</td>
 
                             <td class="success">Basic strain of calcium conversion</td>
−
                             <td class="success">plasmid constructe, molecular cloning</td>
+
                             <td class="success">Plasmid construction, molecular cloning</td>
−
<td class="success">Purchase by Shanghai Weidi Biotechnology</td>
+
                            <td class="success">Purchase by Shanghai Weidi Biotechnology</td>
 
                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
 
                             <td><i>E.coli</i> MG1655</td>
 
                             <td><i>E.coli</i> MG1655</td>
 
                             <td class="success">Type strain</td>
 
                             <td class="success">Type strain</td>
−
                             <td class="success"></td>
+
                             <td class="success">Phenotypic validation</td>
−
                             <td class="success">This work</td>
+
                             <td class="success">Provide by Dr. Chenli Liu (SIAT CSynBER) </td>
 
                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
−
                             <td><i>S. typhimurium</i> SL1344</td>
+
                             <td><i>S.</i> typhimurium SL1344</td>
 
                             <td class="success">Type strain</td>
 
                             <td class="success">Type strain</td>
 
                             <td class="success">Phenotypic validation</td>
 
                             <td class="success">Phenotypic validation</td>
−
                             <td class="success">Provide by Dr.Shan Li （Bio-Medical Center of HZAU</td>
+
                             <td class="success">Provide by Dr. Shan Li (Bio-Medical Center of HZAU)</td>
 
                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
−
                             <td><i>S. typhimurium</i> SL1344(<i>ΔsifA</i>）</td>
+
                             <td><i>S.</i> typhimurium SL1344 (<i>ΔsifA</i>）</td>
 
                             <td class="success">Decrease toxicity and infectivity</td>
 
                             <td class="success">Decrease toxicity and infectivity</td>
 
                             <td class="success">Phenotypic validation</td>
 
                             <td class="success">Phenotypic validation</td>
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                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
−
                             <td><i>S. typhimurium</i> SL1344(<i>ΔsipD</i>）</td>
+
                             <td><i>S.</i> typhimurium SL1344 (<i>ΔsipD</i>）</td>
−
                             <td class="success">knockout Type III secretion system</td>
+
                             <td class="success">Knockout Type III secretion system</td>
 
                             <td class="success">Phenotypic validation</td>
 
                             <td class="success">Phenotypic validation</td>
 
                             <td class="success">This work</td>
 
                             <td class="success">This work</td>
 
                         </tr>
 
                         </tr>
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                 <div class="h2"><b>2.</b>Culture Condition</div>
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                 <div class="h2"><b>2. </b>Culture Condition</div>
 
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                     <thead>
 
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                             <td>Compositions</td>
 
                             <td>Compositions</td>
 
                         </tr>
 
                         </tr>
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+
 
 
                     </thead>
 
                     </thead>
 
                     <tbody>
 
                     <tbody>
 
                         <tr>
 
                         <tr>
−
                             <td>Luria Broth(LB)</td>
+
                             <td>Luria Broth (LB)</td>
−
                             <td>0.5% yeast extraction,1% NaCL and 1% tryptone(add 15 g/L agar when prepare solid culture)</td>
+
                             <td>0.5% yeast extraction,1% NaCl and 1% tryptone (add 15 g/L agar when prepare solid
 +
                                culture)</td>
  
 
                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
−
                             <td>Super Optimal Broth(SOB)</td>
+
                             <td>Super Optimal Broth (SOB)</td>
−
                             <td>0.5% yeast extraction, 0.05% NaCL and 2% tryptone(add 15 g/L agar when prepare solid culture) (add 5ml 2 mol/L MgCl<sub>2</sub> before use.)</td>
+
                             <td>0.5% yeast extraction, 0.05% NaCl and 2% tryptone (add 15 g/L agar when prepare solid
 +
                                culture) (add 5ml 2 mol/L MgCl<sub>2</sub> before use)</td>
 
                         </tr>
 
                         </tr>
 
                     </tbody>
 
                     </tbody>
 
                 </table>
 
                 </table>
 
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             <div id="float03">
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 +
             <div id="float02">
 
                 <div class="h1">Method</div>
 
                 <div class="h1">Method</div>
−
                 <div class="h2"><b>1.</b>Plasmid construction</div>
+
                 <div class="h2"><b>1. </b>Plasmid construction</div>
−
                 <p>Our fragments was PCR amplified with KOD(TOYOBO<sup>®</sup>) or PrimeSTAR(Takara<sup>®</sup>) according to product length. <br><br>
+
                 <p>Our fragments was PCR amplified with KOD (TOYOBO<sup>®</sup>) or PrimeSTAR (Takara<sup>®</sup>)
−
                        Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br><br>
+
                    according to product length. <br><br>
−
                        ClonExpress<sup>®</sup> II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to construct the most plasmids. <br><br>
+
                    Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br><br>
−
                        All plasmid constructs were confirmed by sequencing at Sangon<sup>®</sup>, Inc. (Wuhan,China).<br><br>
+
                    ClonExpress<sup>®</sup> II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to
−
                        A single frozen glycerol stock was used throughout this study for each bacterial strain.<br><br>
+
                    construct the most plasmids. <br><br>
 +
                    All plasmid constructs were confirmed by sequencing at Sangon<sup>®</sup>, Inc. (Wuhan,China).<br><br>
 +
                    A single frozen glycerol stock was used throughout this study for each bacterial strain.<br><br>
 
                 </p>
 
                 </p>
  
−
                 <div class="h2"><b>2.</b>Transformation</div>
+
                 <div class="h2"><b>2. </b>Transformation</div>
 
                 <p>
 
                 <p>
−
                        1. Add 3-5μl plasmid to 50μl F-trans5α(Weidi Biotechnology)competent cells and incubate 5min in ice
+
                    1. Add 3-5μl plasmid to 50μl F-trans5α (Weidi Biotechnology) competent cells and incubate 5min in ice
−
                        , coated plates, select monoclonal colony PCR.Add extra 42℃ heat shock 45s and incubate 10min step will increase transformation efficiency.
+
                    , coated plates, select monoclonal colony PCR. Add extra 42℃ heat shock 45s and incubate 10min step
−
                        Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950rpm for 1h. <br><br>
+
                    will increase transformation efficiency.
−
                      2.Plated on LB agar plates containing appropriate concentration of antibody   for selection, grown overnight at 37°C. And prepare PCR reaction to select individual bacterial colony.
+
                    Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950 rpm for 1h.
 +
                    <br><br>
 +
                    2. Plated on LB agar plates containing appropriate concentration of antibody for selection, grown
 +
                    overnight at 37°C. And prepare PCR reaction to select individual bacterial colony.
 
                 </p>
 
                 </p>
−
                 <div class="h2"><b>3.</b>Fluorescence/growth measurememt</div>
+
                 <div class="h2"><b>3. </b>Fluorescence/growth measurememt</div>
 
                 <p>
 
                 <p>
−
                        Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is 0.1 with fresh LB broth.<br><br>
+
                    Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is
−
                        Expression was induced at early log phase by addition of different atc (40–140 ng/ml) concentrations. Culture the plate in 37℃ , 150 rpm. Every hour put it into a plate reader to measure its fluorescence/OD<sup>1</sup>.
+
                    0.1 with fresh LB broth.<br><br>
 +
                    Expression was induced at early log phase by addition of different ATc (150 ng/ml–15μg/ml)
 +
                    concentrations. Culture the plate in 37℃, 150 rpm. Every hour put it into a plate reader to
 +
                    measure its fluorescence/OD<sup>1</sup>.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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             <div id="float03">
 
                 <div class="h1">Modeling impact our experiment</div>
 
                 <div class="h1">Modeling impact our experiment</div>
−
                 <p>The higher ATc level might thwarts bacterial growth.So we need to analyze a proper concentration of ATc, We design a modeling to predict a suitable concentration of ATc(More result about our model please check <a href="https://2018.igem.org/Team:HZAU-China/Model">modeling</a>). We induced our engineered strain with gradients of aTc,.Take 100μl culture to plate on LB agar plates containing appropriate concentration of antibody after 5 minutes, 30 minutes, 90 minutes of induce and counting Colony Forming Units the next day. The result fit the model within a range of ATc. So we choose a appropriate concentration of ATc(16μg/ml), aiming to maximize the expression of GSDMD-N275 and minimize the negative effect of bacteria growth.      
+
                 <p>The higher ATc level might thwart bacterial growth. So we need to analyze a proper concentration of
 +
                    ATc, We design a modeling to predict a suitable concentration of ATc (More result about our model
 +
                    please check <a href="https://2018.igem.org/Team:HZAU-China/Model">modeling: Chemical control model</a>).
 +
                    We induced our engineered strain with gradients of ATc. Take 100μl culture to plate on LB agar
 +
                    plates containing appropriate concentration of antibody after 5 minutes, 30 minutes, 90 minutes of
 +
                    induce and counting Colony Forming Units the next day. The results fit the model within a range of
 +
                    ATc. So we choose a appropriate concentration of ATc (16μg/ml), aiming to maximize the expression of
 +
                    GSDMD-N275 and minimize the negative effect of bacteria growth.
 
                 </p>
 
                 </p>
 
                 <div class="h1">Reference</div>
 
                 <div class="h1">Reference</div>
−
                 <p>1.Becskel, A. & Serrano, L. Engineering stability in gene networks by autoregulation. Nature 405, 590–593 (2000).</p>
+
                 <p>1. Becskel, A. & Serrano, L. Engineering stability in gene networks by autoregulation. Nature 405,
 +
                    590–593 (2000).</p>
 
             </div>
 
             </div>
 +
        </div>
 
     </div>
 
     </div>
−
</div>
 
  
−
        <!-- 侧边 -->
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     <div class="cebian">
 
     <div class="cebian">
 
         <!-- 滚动菜单栏 -->
 
         <!-- 滚动菜单栏 -->
 
         <div class="daohangyi">
 
         <div class="daohangyi">
−
            <span class="biaoti">Safety</span>
+
                <span class="biaoti">Experiments</span>
−
            <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg" alt=""></span>
+
                <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/8/8d/T--HZAU-China--xjt.svg" alt=""></span>
 
         </div>
 
         </div>
 
         <div class="floatCtro">
 
         <div class="floatCtro">
−
             <p class="daohanger">Introduction</p>
+
             <p class="daohanger">Material</p>
−
             <p class="daohanger">Lab Safety</p>
+
             <p class="daohanger">Method</p>
 +
            <p class="daohanger">Modeling impact our experiment</p>
 
             <a>
 
             <a>
 
                 <span>Back&nbsp;to&nbsp;Top</span>
 
                 <span>Back&nbsp;to&nbsp;Top</span>
 
             </a>
 
             </a>
 
         </div>
 
         </div>
−
         <a class="daohangyi" href="https://2018.igem.org/Team:HZAU-China/Human_Practices">
+
         <a class="daohangyi" href="https://2018.igem.org/Team:HZAU-China/Improve">
−
             <span class="biaoti">Human Practices</span>
+
             <span class="biaoti">Improve</span>
 +
            <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg" alt=""></span>
 +
        </a>
 +
        <a class="daohangyi" href="https://2018.igem.org/Team:HZAU-China/InterLab">
 +
            <span class="biaoti">InterLab</span>
 
             <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg" alt=""></span>
 
             <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg" alt=""></span>
 
         </a>
 
         </a>
−
         <a class="daohangyi" href="https://2018.igem.org/Team:HZAU-China/Public_Engagement">
+
         <a class="daohangyi" href="https://2018.igem.org/Team:HZAU-China/Notebook">
−
             <span class="biaoti">Public Engagement</span>
+
             <span class="biaoti">Notebook</span>
 
             <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg" alt=""></span>
 
             <span class="xsjPic"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--HZAU-China--ysj.svg" alt=""></span>
 
         </a>
 
         </a>

Latest revision as of 03:57, 18 October 2018

Material
1. Bacteria Strains used in this work
Strain Description Usage Sourse
E.coli trans-5α Basic strain of calcium conversion Plasmid construction, molecular cloning Purchase by Shanghai Weidi Biotechnology
E.coli MG1655 Type strain Phenotypic validation Provide by Dr. Chenli Liu (SIAT CSynBER)
S. typhimurium SL1344 Type strain Phenotypic validation Provide by Dr. Shan Li (Bio-Medical Center of HZAU)
S. typhimurium SL1344 (ΔsifA） Decrease toxicity and infectivity Phenotypic validation This work
S. typhimurium SL1344 (ΔsipD） Knockout Type III secretion system Phenotypic validation This work
2. Culture Condition
Culture medium Compositions
Luria Broth (LB) 0.5% yeast extraction,1% NaCl and 1% tryptone (add 15 g/L agar when prepare solid culture)
Super Optimal Broth (SOB) 0.5% yeast extraction, 0.05% NaCl and 2% tryptone (add 15 g/L agar when prepare solid culture) (add 5ml 2 mol/L MgCl2 before use)
Method
1. Plasmid construction

Our fragments was PCR amplified with KOD (TOYOBO®) or PrimeSTAR (Takara®) according to product length.

Product is recycled with gel extraction kit from OMEGA® after electrophoresis.

ClonExpress® II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to construct the most plasmids.

All plasmid constructs were confirmed by sequencing at Sangon®, Inc. (Wuhan,China).

A single frozen glycerol stock was used throughout this study for each bacterial strain.

2. Transformation

1. Add 3-5μl plasmid to 50μl F-trans5α (Weidi Biotechnology) competent cells and incubate 5min in ice , coated plates, select monoclonal colony PCR. Add extra 42℃ heat shock 45s and incubate 10min step will increase transformation efficiency. Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950 rpm for 1h.

2. Plated on LB agar plates containing appropriate concentration of antibody for selection, grown overnight at 37°C. And prepare PCR reaction to select individual bacterial colony.

3. Fluorescence/growth measurememt

Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is 0.1 with fresh LB broth.

Expression was induced at early log phase by addition of different ATc (150 ng/ml–15μg/ml) concentrations. Culture the plate in 37℃, 150 rpm. Every hour put it into a plate reader to measure its fluorescence/OD1.

Modeling impact our experiment

The higher ATc level might thwart bacterial growth. So we need to analyze a proper concentration of ATc, We design a modeling to predict a suitable concentration of ATc (More result about our model please check modeling: Chemical control model). We induced our engineered strain with gradients of ATc. Take 100μl culture to plate on LB agar plates containing appropriate concentration of antibody after 5 minutes, 30 minutes, 90 minutes of induce and counting Colony Forming Units the next day. The results fit the model within a range of ATc. So we choose a appropriate concentration of ATc (16μg/ml), aiming to maximize the expression of GSDMD-N275 and minimize the negative effect of bacteria growth.

Reference

1. Becskel, A. & Serrano, L. Engineering stability in gene networks by autoregulation. Nature 405, 590–593 (2000).

Experiments

Material

Method

Modeling impact our experiment

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