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>
 
                 <table class="table table-bordered table-hover">
 
                 <table class="table table-bordered table-hover">
 
                     <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>
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                             <td class="success">plasmid constructe, molecular cloning</td>
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                             <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>
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                             <td class="success">Type strain</td>
 
                             <td class="success">Type strain</td>
 
                             <td class="success">Phenotypic validation</td>
 
                             <td class="success">Phenotypic validation</td>
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                             <td class="success">Provide by Dr.Shan Li (Bio-Medical Center of HZAU)</td>
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                             <td class="success">Provide by Dr. Shan Li (Bio-Medical Center of HZAU)</td>
 
                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
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                         <tr>
 
                         <tr>
 
                             <td><i>S.</i> typhimurium SL1344 (<i>ΔsipD</i>）</td>
 
                             <td><i>S.</i> typhimurium SL1344 (<i>ΔsipD</i>）</td>
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                             <td class="success">knockout Type III secretion system</td>
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                             <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>
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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>
 
                 <table class="table table-bordered table-hover">
 
                 <table class="table table-bordered table-hover">
 
                     <thead>
 
                     <thead>
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                             <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
 
                             <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>
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                                 culture) (add 5ml 2 mol/L MgCl<sub>2</sub> before use)</td>
 
                         </tr>
 
                         </tr>
 
                     </tbody>
 
                     </tbody>
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             <div id="float02">
 
             <div id="float02">
 
                 <div class="h1">Method</div>
 
                 <div class="h1">Method</div>
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                 <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>)
 
                 <p>Our fragments was PCR amplified with KOD (TOYOBO<sup>®</sup>) or PrimeSTAR (Takara<sup>®</sup>)
 
                     according to product length. <br><br>
 
                     according to product length. <br><br>
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                 </p>
 
                 </p>
  
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                 <div class="h2"><b>2.</b>Transformation</div>
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                 <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
 
                     , coated plates, select monoclonal colony PCR. Add extra 42℃ heat shock 45s and incubate 10min step
 
                     will increase transformation efficiency.
 
                     will increase transformation efficiency.
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                     Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950rpm for 1h.
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                     Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950 rpm for 1h.
 
                     <br><br>
 
                     <br><br>
 
                     2. Plated on LB agar plates containing appropriate concentration of antibody for selection, grown
 
                     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.
 
                     overnight at 37°C. And prepare PCR reaction to select individual bacterial colony.
 
                 </p>
 
                 </p>
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                 <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
 
                     Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is
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             <div id="float03">
 
             <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
+
                 <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
 
                     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>).
+
                     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
 
                     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
 
                     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
+
                     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
 
                     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.
 
                     GSDMD-N275 and minimize the negative effect of bacteria growth.

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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