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

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                 <div class="h1">Material</div>
 
                 <div class="h1">Material</div>
 
                  
 
                  
−
                 <div class="h2"><b>1.</b>>Bacteria Strains used in this work</div>
+
                 <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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                         </tr>
 
                         </tr>
 
                         <tr>
 
                         <tr>
−
                             <td><i>S. typhimurium</i>> SL1344</td>
+
                             <td><i>S. typhimurium</i> 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. typhimurium</i> 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. typhimurium</i> 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="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>
+
                 <p>Our fragments was PCR amplified with KOD(TOYOBO<sup>®</sup>) or PrimeSTAR(Takara<sup>®</sup>) according to product length. <br><br>
−
                         Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br>
+
                         Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br><br>
−
                         ClonExpress<sup>®</sup> II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to construct the most plasmids. <br>
+
                         ClonExpress<sup>®</sup> II One Step Cloning Kit (Vyzyme) was used to ligate every fragment to construct the most plasmids. <br><br>
−
                         All plasmid constructs were confirmed by sequencing at Sangon<sup>®</sup>, Inc. (Wuhan,China).<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>
+
                         A single frozen glycerol stock was used throughout this study for each bacterial strain.<br><br>
 
                 </p>
 
                 </p>
  
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                         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 will increase  transformation efficiency.
−
                         Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950rpm for 1h. <br>
+
                         Or 1.5kv, 4-5ms electroporate cell with 300-400 ng purified plasmid. Recover at 37°C 950rpm 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.
 
                       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>
+
                         Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted to OD is 0.1 with fresh LB broth.<br><br>
 
                         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>.
 
                         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>.
 
                 </p>
 
                 </p>

Revision as of 05:10, 17 October 2018

Material
1.Bacteria Strains used in this work
Strain Description Usage Sourse
E.coli trans-5α Basic strain of calcium conversion plasmid constructe, molecular cloning Purchase by Shanghai Weidi Biotechnology
E.coli MG1655 Type strain This work
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 950rpm 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 (40–140 ng/ml) concentrations. Culture the plate in 37℃ , 150 rpm. Every hour put it into a plate reader to measure its fluorescence/OD1.

Reference

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

Safety

Introduction

Lab Safety

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