Difference between revisions of "Team:HZAU-China/Experiments"
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| Line 744: | Line 744: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><i>S. | + | <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> | ||
| Line 750: | Line 750: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><i>S. | + | <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> | ||
| Line 756: | Line 756: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td><i>S. | + | <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> | ||
| Line 780: | Line 780: | ||
<tr> | <tr> | ||
<td>Luria Broth(LB)</td> | <td>Luria Broth(LB)</td> | ||
| − | <td>0.5% yeast extraction,1% | + | <td>0.5% yeast extraction,1% NaCl and 1% tryptone(add 15 g/L agar when prepare solid |
culture)</td> | culture)</td> | ||
| Line 786: | Line 786: | ||
<tr> | <tr> | ||
<td>Super Optimal Broth(SOB)</td> | <td>Super Optimal Broth(SOB)</td> | ||
| − | <td>0.5% yeast extraction, 0.05% | + | <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> | culture) (add 5ml 2 mol/L MgCl<sub>2</sub> before use.)</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>) | + | <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> | ||
Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br><br> | Product is recycled with gel extraction kit from OMEGA<sup>®</sup> after electrophoresis. <br><br> | ||
| Line 810: | Line 810: | ||
<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 | , coated plates, select monoclonal colony PCR. Add extra 42℃ heat shock 45s and incubate 10min step | ||
will increase transformation efficiency. | will increase transformation efficiency. | ||
| Line 823: | Line 823: | ||
0.1 with fresh LB broth.<br><br> | 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) | 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 | + | concentrations. Culture the plate in 37℃, 150 rpm. Every hour put it into a plate reader to |
measure its fluorescence/OD<sup>1</sup>. | measure its fluorescence/OD<sup>1</sup>. | ||
</p> | </p> | ||
Revision as of 20:21, 17 October 2018
| 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 | 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 |
| 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.) |
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.
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.
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.
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 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 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.
1. Becskel, A. & Serrano, L. Engineering stability in gene networks by autoregulation. Nature 405, 590–593 (2000).
