|
|
| Line 19: |
Line 19: |
| | <h4>Taq:</h4> | | <h4>Taq:</h4> |
| | <figure class="text-center"> | | <figure class="text-center"> |
| − | <img src="https://static.igem.org/mediawiki/2018/3/32/T--BNU-China--image_protocal_1.jpg" width="100%"> | + | <img src="https://static.igem.org/mediawiki/2018/3/32/T--BNU-China--image_protocal_1.jpg" width="100%"> |
| − | <figcaption>
| + | <figcaption> |
| − | Fig.1 Degradation of engineered bacteria
| + | </figcaption> |
| − | </figcaption>
| + | |
| | </figure> | | </figure> |
| | + | <h4>Super mix:</h4> |
| | + | <figure class="text-center"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/2/2a/T--BNU-China--image_protocal2.jpg" width="100%"> |
| | + | <figcaption> |
| | + | </figcaption> |
| | + | </figure> |
| | + | <h3>2) 50μL system for PCR target genes</h3> |
| | + | <h4>Pfu mix:</h4></br> |
| | + | <h4>DNA: plasmid 50ng; genome 100~200ng</h4> |
| | + | <figure class="text-center"> |
| | + | <img src=" https://static.igem.org/mediawiki/2018/c/cd/T--BNU-China--image_p3.jpg" width="100%"> |
| | + | <figcaption> |
| | + | </figcaption> |
| | + | </figure> |
| | + | <h3>3) 20μL PCR system for microbes</h3> |
| | + | <h4>Super mix:</h4></br><h4>Bacteria (after picking on the board, back up on the new board and rinse it in the system that has been prepared)</h4> |
| | + | <figure class="text-center"> |
| | + | <img src="https://static.igem.org/mediawiki/2018/a/a4/T--BNU-China--image_p4.jpg" width="100%"> |
| | + | <figcaption> |
| | + | </figcaption> |
| | + | </figure> |
| | + | |
| | + | |
| | | | |
| − | <h4>Fusion PCR：</h4>
| |
| − | <ol>
| |
| − | <li> basic PCR</li>
| |
| − | <li> use the PCR product of step 1 as template to do PCR</li>
| |
| − | <li> use the PCR product of step 2 as template to do PCR, but first five cycles don’t add primer,
| |
| − | add primer at the sixth cycle and continue PCR.
| |
| − | </li>
| |
| − | </ol>
| |
| − | <h5> The system of step 2: </h5>
| |
| − | <p>H<sub>2</sub>O 21μL</p>
| |
| − | <p>2x primeSTAR 25mL</p>
| |
| − | <p>R+F-Primer 2 μL</p>
| |
| − | <p>Template① 1μL</p>
| |
| − | <p>Template② 1μL</p>
| |
| − |
| |
| − | <h3>Electrophoresis---Gel Purification</h3>
| |
| − | <h4>Material:</h4>
| |
| − | <p>Agarose gel: 1% agarose dissolved in 1 x TAE + gelstain</p>
| |
| − | <h4>Protocol:</h4>
| |
| − | <p>We used gelstain to stain the DNA and imaged it in a Transilluminator.</p>
| |
| − | <p>We used the gel extraction kit to get the objective fragment.</p>
| |
| − | <p>We used the DNA fragment purification kit to get the objective fragment.</p>
| |
| − | <h3>Digestion</h3>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="6" style="text-align: center">50 μL reaction system</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
| |
| − | <td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
| |
| − | <td align="center">\(Pst\ \mathrm{I}\)</td>
| |
| − | <td align="center">\(\mathrm{Plasmid}\)</td>
| |
| − | <td align="center">\(\mathrm{H_2 O}\)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">5 μL</td>
| |
| − | <td align="center">1.5 μL</td>
| |
| − | <td align="center">1.5 μL</td>
| |
| − | <td align="center">15 μL</td>
| |
| − | <td align="center">27 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="6" style="text-align: center">10 μL reaction system</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">10x \(\ \mathrm{H \ buffer}\)</td>
| |
| − | <td align="center">\(Eco\mathrm{R}\ \mathrm{I}\)</td>
| |
| − | <td align="center">\(Pst\ \mathrm{I}\)</td>
| |
| − | <td align="center">\(\mathrm{Plasmid}\)</td>
| |
| − | <td align="center">\(\mathrm{H_2 O}\)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">1 μL</td>
| |
| − | <td align="center">0.3 μL</td>
| |
| − | <td align="center">0.3 μL</td>
| |
| − | <td align="center">3 μL</td>
| |
| − | <td align="center">5.4 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h2>Ligation</h2>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="5" style="text-align: center">Ligation reaction system</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">DNA</td>
| |
| − | <td align="center">Plasmid</td>
| |
| − | <td align="center">T4 buffer</td>
| |
| − | <td align="center">T4 ligase</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">7 μL</td>
| |
| − | <td align="center">1 μL</td>
| |
| − | <td align="center">1 μL</td>
| |
| − | <td align="center">1 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h3>LR reaction</h3>
| |
| − |
| |
| − | <h4>1. Entry linearization</h4>
| |
| − | <p>β2-TOPO (plasmid concentration 117 ng/μL)NotI 37°C enzyme digestion for the night</p>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="2"> 50 μL Single enzyme system</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">10x BufferH</td>
| |
| − | <td align="center">5 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">DNA</td>
| |
| − | <td align="center">20 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">ddH<sub>2</sub>O</td>
| |
| − | <td align="center">12.5 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Enzyme</td>
| |
| − | <td align="center">2.5 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">0.1%BSA</td>
| |
| − | <td align="center">5 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">0.1%Triton X-100</td>
| |
| − | <td align="center">5 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h4>2. LR system (\(4\mu\mathrm{L}\)):</h4>
| |
| − | <p>100 ng/μL linear Entry: 0.5 μL</p>
| |
| − | <p>destination vector: 1 μL (pCambia1300-nluc / pCambia1300-cluceach one)</p>
| |
| − | <p>LR Clonase II enzyme mix: 1 μL</p>
| |
| − | <p>ddH<sub>2</sub>O: 0.5 μL</p>
| |
| − | <p>mix slightly, water base for 5h at 25°C </p>
| |
| − | <p>transform, 4 μL, reactant transform 50 μL competent cells</p>
| |
| − |
| |
| − | <h2>Transformation</h2>
| |
| − |
| |
| − | <h3>Material:</h3>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="4" style="text-align: center">LB liquid medium</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">Tryptone</td>
| |
| − | <td align="center">Yeast extract powder</td>
| |
| − | <td align="center">NaCl</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">10 g/L</td>
| |
| − | <td align="center">5 g/L</td>
| |
| − | <td align="center">10 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h3>Protocol:</h3>
| |
| − | <p> Preparation of the competent cells </p>
| |
| − | <p> 1 μL ligation product + 50 μL cells </p>
| |
| − | <p>Heatshock of Trans5α(42°C, 45s)</p>
| |
| − | <p>Put on ice(2min)</p>
| |
| − | <p>Add 500 μL LB media and incubate for 1h(37°C, 150rpm)</p>
| |
| − | <p>Centrifuge at 4000 rpm for 1min and remove 400 μL supernatant</p>
| |
| − | <p>Resuspend the pellets using the left supernatant</p>
| |
| − | <p>Spread plates(with Kan;Chl)</p>
| |
| − | <p>Incubate for 12~16h(37°C)</p>
| |
| − |
| |
| − | <h2>Protein expression</h2>
| |
| − | <ol>
| |
| − | <li>Inoculated 3 mL LB media including relevant antibiotics with the monoclonal colony of expression
| |
| − | plasmid, incubate for 12~16h(37°C, 190rpm)
| |
| − | </li>
| |
| − | <li>Inoculated 100 mL TM expression media including relevant antibiotics with the 1 mL bacteria
| |
| − | liquid, incubate for 3h(37°C, 250rpm,OD<sub>600</sub>=0.6~0.8)
| |
| − | </li>
| |
| − | <li>Add IPTG into it until its final concentration is 1 mmol/L, incubate for 4~6h(37°C,
| |
| − | 250rpm)
| |
| − | </li>
| |
| − | <li>Centrifuge at 6000 rpm for 10min and remove supernatant</li>
| |
| − | <li>Gather sediment, cryopreserve at -20°C</li>
| |
| − | </ol>
| |
| − | <p>Material:</p>
| |
| − | <p>TM expression medium:1000 mL pH=7.4</p>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">tryptone</td>
| |
| − | <td align="center">Yeast extract powder</td>
| |
| − | <td align="center">NaCl</td>
| |
| − | <td align="center">glucose</td>
| |
| − | <td align="center">glycerol</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">1.2 g</td>
| |
| − | <td align="center">2.4 g</td>
| |
| − | <td align="center">1.0 g</td>
| |
| − | <td align="center">1.0 g</td>
| |
| − | <td align="center">0.6 mL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <p>Autoclaving 115℃, 20min</p>
| |
| − |
| |
| − | <h2>Detection</h2>
| |
| − | <h3>SDS-PAGE</h3>
| |
| − | <h4>Materials</h4>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center">Gel</th>
| |
| − | <th style="text-align: center">Tris-HCl</th>
| |
| − | <th style="text-align: center">Acr/Bis 30%</th>
| |
| − | <th style="text-align: center">SDS 10%</th>
| |
| − | <th style="text-align: center">ddH<sub>2</sub>O</th>
| |
| − | <th style="text-align: center">TEMED</th>
| |
| − | <th style="text-align: center">AP 10%</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Stacking Gel(4%)</td>
| |
| − | <td align="center">pH=6.8 500 μL</td>
| |
| − | <td align="center">500 μL</td>
| |
| − | <td align="center">25 μL</td>
| |
| − | <td align="center">1350 μL</td>
| |
| − | <td align="center">2.5 μL</td>
| |
| − | <td align="center">12.5 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Running Gel(12%)</td>
| |
| − | <td align="center">pH=8.8 1250 μL</td>
| |
| − | <td align="center">2000 μL</td>
| |
| − | <td align="center">50 μL</td>
| |
| − | <td align="center">1675 μL</td>
| |
| − | <td align="center">2.5 μL</td>
| |
| − | <td align="center">25 μL</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Running Gel(18%)</td>
| |
| − | <td align="center">pH=8.8 1250 μL</td>
| |
| − | <td align="center">3000 μL</td>
| |
| − | <td align="center">50 μL</td>
| |
| − | <td align="center">675 μL</td>
| |
| − | <td align="center">2.5 μL</td>
| |
| − | <td align="center">25 μL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="4" style="text-align: center">Running Buffer</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">Tris-HCl</td>
| |
| − | <td align="center">Glycine</td>
| |
| − | <td align="center">(w/v) SDS</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">25 mmol/L</td>
| |
| − | <td align="center">0.192 mol/L</td>
| |
| − | <td align="center">0.1%</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <h4>Protocol</h4>
| |
| − | <p>The SDS polyacrylamide gels are prepared in the so-called PerfectBlue™ Twin Double Gel System.</p>
| |
| − | <p>After ensuring that the equipment is waterproof, the 12% (or 18%) running gel is mixed and filled
| |
| − | into the chamber. Pipetting about 1 ml of H<sub>2</sub>O on top of the running gel to seal the gel.
| |
| − | </p>
| |
| − | <p>After polymerization, the remaining H<sub>2</sub>O is removed and the 12% stacking gel is filled on
| |
| − | top. Insert
| |
| − | a comb to create sample pockets.</p>
| |
| − | <p>After the stacking gel also polymerized, 1 x running buffer is used to run the Double Gel System via
| |
| − | the SDS gel. </p>
| |
| − | <p>After loading the generated pockets with the samples, the stacking gel is run at 100 V and then
| |
| − | running gel at 120 V. </p>
| |
| − |
| |
| − | <h3>Western Blot</h3>
| |
| − | <h4>System</h4>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="6" style="text-align: center"> PBST:1000 mL(pH=7.4)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">NaCl(137mM)</td>
| |
| − | <td align="center">KCl(2.7mM)</td>
| |
| − | <td align="center">Na<sub>2</sub>HPO<sub>4</sub>(10mM)</td>
| |
| − | <td align="center">K<sub>2</sub>HPO<sub>4</sub>(2mM)</td>
| |
| − | <td align="center">Tween-20</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">8 g</td>
| |
| − | <td align="center">0.2 g</td>
| |
| − | <td align="center">1.44 g</td>
| |
| − | <td align="center">0.24 g</td>
| |
| − | <td align="center">0.5 mL</td>
| |
| − | </tr>
| |
| − |
| |
| − | </table>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th colspan="4" style="text-align: center">Imprint buffer：2000 mL (pH=8.3) Transfer Buffer</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">Tris</td>
| |
| − | <td align="center">Gly</td>
| |
| − | <td align="center">Methanol</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">6.06 g</td>
| |
| − | <td align="center">28.8 g</td>
| |
| − | <td align="center">400 mL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h4>Protocol</h4>
| |
| − | <p>Transfer (Prepare transfer Buffer just before glue leaking, and precool at -20℃).</p>
| |
| − | <ol>
| |
| − | <li>Put the transfer Buffer and the black subface of transfer splint downword, and lay a sponge in
| |
| − | it. Several filter paper(three pieces of filter paper), glue(except Stacking Gel).
| |
| − | </li>
| |
| − | <li>Activation PVDF membrane in advance with anhydrous ethanol, and put it on the membrane.</li>
| |
| − | <li>Three layers of filter paper, sponge, Squeeze out of the bubbles, turn tight.</li>
| |
| − | <li>The black subface electric rotary groove stick to each other, put in ice.</li>
| |
| − | <li>110V, 120min.</li>
| |
| − | <li>5% skim milk powder (prepared by PBST), block for a night.</li>
| |
| − | <li>Dilute Primary antibody at the proportion of 1:2000 with 3% skim milk powder(add 0.02% sodium
| |
| − | azide ), incubate 1h at the room temperature.
| |
| − | </li>
| |
| − | <li>PBST elute, wash with shocking for 5min , three times.</li>
| |
| − | <li>Dilute Secondary antibody at the proportion of 1:2000 with 3% skim milk powder, incubate 1h at
| |
| − | the room temperature.
| |
| − | </li>
| |
| − | <li>PBST elute, wash with shocking for 5min, three times.</li>
| |
| − | <li>Color development.</li>
| |
| − | </ol>
| |
| − |
| |
| − | <h2>Ni-beads protein purification</h2>
| |
| − |
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="4"> NPI-10 buffer(1L) pH=8.0 filtration sterilization
| |
| − | </th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
| |
| − | <td align="center">NaCl</td>
| |
| − | <td align="center">imidazole</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">6.9</td>
| |
| − | <td align="center">17.54</td>
| |
| − | <td align="center">0.68</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="4">NPI-20 buffer(1L) pH=8.0 filtration sterilization</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
| |
| − | <td align="center">NaCl</td>
| |
| − | <td align="center">imidazole</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage(g)</td>
| |
| − | <td align="center">6.9</td>
| |
| − | <td align="center">17.54</td>
| |
| − | <td align="center">1.36</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="4">NPI-250 buffer(1L) pH=8.0 filtration sterilization
| |
| − | </th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">NaH<sub>2</sub>PO<sub>4</sub>·H<sub>2</sub>O</td>
| |
| − | <td align="center">NaCl</td>
| |
| − | <td align="center">imidazole</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage (g)</td>
| |
| − | <td align="center">6.9</td>
| |
| − | <td align="center">17.54</td>
| |
| − | <td align="center">17.0</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <h4>Protocol</h4>
| |
| − | <ol>
| |
| − | <li>Cut tips. Add 30 μL Ni 6 fast flow Beads into 1.5 mL EP.</li>
| |
| − | <li>Add 1 mL NPI-10 buffer, mixing wash, sedimentate at low speed and wash 3 times.</li>
| |
| − | <li>Centrifuge and absorb supernatant into buffer. 4℃ binding 3h, rotate and mix.</li>
| |
| − | <li>After binding, Put on ice(5min), Centrifuge at 2000rpm for 1min.</li>
| |
| − | <li>Absorb 80μL supernatant as control and remove the other supernatant, add 1 mL NPI-20 washing,
| |
| − | upside and down to mix, still standing, Centrifuge at 2000 rpm for 1min(4℃), wash 3~5 times.
| |
| − | </li>
| |
| − | <li>Add 500μL NPI-250 into Beads, rotate and mix for 15min, gather supernatant, add 500 μL NPI-250 ,
| |
| − | rotate and mix for 15min, gather supernatant again.
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h2>Renaturation of the inclusion bodies</h2>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="4">Binding buffer(1L) pH=8.0</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">NaCl(500 mmol/L)</td>
| |
| − | <td align="center">Na<sub>3</sub>PO<sub>4</sub>·12H<sub>2</sub>O(20 mmol/L)</td>
| |
| − | <td align="center">imidazole (20 mmol/L)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage(g)</td>
| |
| − | <td align="center">29.22</td>
| |
| − | <td align="center">7.6</td>
| |
| − | <td align="center">1.36</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <p>After cell disruption, sediment dissolves in binding buffer(8 mol/L urea)</p>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="5">Washing buffer(1 L)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Reagent</td>
| |
| − | <td align="center">Tris-HCl(50 mmol/L)</td>
| |
| − | <td align="center">EDTA(5 mmol/L)</td>
| |
| − | <td align="center">NaCl(100 mmol/L)</td>
| |
| − | <td align="center">Triton X-100(1%)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">100 mL</td>
| |
| − | <td align="center">1.8612 g</td>
| |
| − | <td align="center">5.844 g</td>
| |
| − | <td align="center">10 mL</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <ol>
| |
| − | <li>Induced expression</li>
| |
| − | <li>Collect sediment after ultrasonication, use washing buffer including 2, 3 mol/L urea to wash
| |
| − | sediment in turn. Finally, use washing buffer including 8 mol/L urea to dissolve. Centrifuge and
| |
| − | absorb supernatant, measure protein concentration and make it keep about 1 mg/mL. Dialyze these
| |
| − | supernatant using binding buffer including concentration gradient urea(6,5,4,3,2,1,0.5 and
| |
| − | 0 mol/L).
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − |
| |
| − | <h2>Extracting tubulin from porcine brains</h2>
| |
| − | <ol>
| |
| − | <li>Pick up 20 porcine brains from Beijing No.5 Meat Processing. For tubulin extraction
| |
| − | experiment, the brains should be as fresh as possible. Take an ice box to store the brains.
| |
| − | Avoid contact between the brains.
| |
| − | </li>
| |
| − | <li>While getting the brains, another student should stay in the lab and prepared the centrifuge
| |
| − | (set one at 4℃ and another at 37℃, also pre-warm the rotor). Put electronic balance, grinder, a
| |
| − | graduated cylinder in refrigerator and pre-warm the glycerol and PEM in 37℃. prepare the fresh
| |
| − | ATP and GTP buffer in the morning.
| |
| − | </li>
| |
| − | <li>Clean the brain by tearing off the meninges and blood clots using kim wipes or by hand.</li>
| |
| − | <li>After cleaning, weigh the brains, put the brains in the blender, then add the same volume buffer
| |
| − | PEM(with 1 mM DTT) in it accordingly.
| |
| − | </li>
| |
| − | <li>Homogenate the brain for 3s, 10 times, time interval between two homogenate is 5s, in order to
| |
| − | avoid destroy the tubulin because of high thermos.
| |
| − | </li>
| |
| − | <li>Pour the homogenate into a flask, incubate in 4℃ for 30 min to depolymerize microtubules.</li>
| |
| − | <li>Pour the homogenate into tubes for Type 45Ti rotor and balance each tube.</li>
| |
| − | <li>Centrifuge at 8000 rpm for 40min at 4℃. Filter the supernatant with 4 gauzes. Then centrifuge
| |
| − | the filtrate at 40000 g for 40min at 4℃.
| |
| − | </li>
| |
| − | <li>Add 1/2 volume of warmed glycerol drop-wise with continuous shaking, mix gently but thoroughly.
| |
| − | Add GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final concentration 3 mmol/L), and
| |
| − | EGTA (final
| |
| − | concentration 1 mmol/L). Incubate in a 37℃ water bath for 1h, shake gently and occasionally.
| |
| − | </li>
| |
| − | <li>Balance each tube and centrifuge at 100000 g for 40 min at 35℃.Discard supernatant, the pellet
| |
| − | is crude extracts. Split charge them into 50 mL centrifuge tubes, each tube contains 5 g. Snap
| |
| − | freeze the tubulin in 15 μL aliquots in liquid nitrogen and further stored at -80℃.
| |
| − | </li>
| |
| − | <li>When going to do refined depuration, melt the freezing crude extract at 4℃ refrigerator on ice
| |
| − | over night. Pop out 1 g of the pellet out of the tubes with a spatula. Put the pellets in the
| |
| − | Dounce grinder, then add cold PEM in the tube to wash off residual pellets.
| |
| − | </li>
| |
| − | <li>Re-suspend the pellets with grinder, keep the grinder on ice, and grinding occasionally. After
| |
| − | 30min, pour out the solution and rinse the grinder with cold PEM. Total re-suspended volume is
| |
| − | 5 mL.
| |
| − | </li>
| |
| − | <li>Add GTP (final concentration 0.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it
| |
| − | occasionally.
| |
| − | </li>
| |
| − | <li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.</li>
| |
| − | <li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES( pH =6.9 ),
| |
| − | DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final
| |
| − | concentration
| |
| − | 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
| |
| − | </li>
| |
| − | <li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
| |
| − | <li>Balance each tube and centrifuge at 100000 g for 1h at 35℃.</li>
| |
| − | <li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
| |
| − | 0.1 mmol/L). Place it on ice for 1h to depolymerize. Shake it occasionally.
| |
| − | </li>
| |
| − | <li>Centrifuge the depolymerized tubulin at 100000 g for 40min at 4 ℃.</li>
| |
| − | <li>Recover the supernatant and pour it into a flask. Add equal volume of warmed PIPES ( pH= 6.9 ),
| |
| − | DMSO(final concentration 10%), GTP (final concentration 0.1 mmol/L), MgCl<sub>2</sub> (final
| |
| − | concentration 1 mmol/L) and EGTA (final concentration 1 mmol/L). Mix gently but thoroughly.
| |
| − | </li>
| |
| − | <li>Incubate in a 37℃ water bath for 1 h. the solution would look cloudy.</li>
| |
| − | <li>Balance each tube and centrifuge at 100000 g for 1h at 35℃.</li>
| |
| − | <li>Discard the supernatant. Risen the pellet briefly with cold PEM, add GTP (final concentration
| |
| − | 0.1 mmol/L). Place it on ice for 1 h to depolymerize. Shake it occasionally.
| |
| − | </li>
| |
| − | <li>Centrifuge the depolymerized tubulin at 100000 g for 40 min at 4℃.</li>
| |
| − | <li>Recover the supernatant, add equal volume polymerize buffer (containing 100 mmol/L PIPES-KOH, 2
| |
| − | mmol/L EGTA, 2 mmol/L MgCl<sub>2</sub>, 2 mmol/L GTP and 60% glycerol).
| |
| − | </li>
| |
| − | <li>Incubate in a 37℃ water bath for 1 h. Adding a series concentrations of taxol.</li>
| |
| − | <li>Balance each tube and centrifuge at 100000 g for 1 h at 35℃. The pellet is fine purified
| |
| − | product.
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>Preparing STM samples</h3>
| |
| − | <ol>
| |
| − | <li>Use 200 # copper mesh to prepare the samples. This procedure was done in Institute of
| |
| − | Biophysics, Chinese Academy of Science. Pretreat the copper mesh with Varian Plasma Cleaner PDC-32G.</li>
| |
| − | <li>Add protein solution on copper mesh. Keep still for 1 min.</li>
| |
| − | <li>Add 3 drops of uranyl acetate on parafilm. Mix the protein binding side with uranyl acetate
| |
| − | thoroughly.
| |
| − | </li>
| |
| − | <li>Store the sample carefully. Observe by transmission electron microscopy in Beijing Normal
| |
| − | University.
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h3> OD<sub>350</sub> test </h3>
| |
| − | <ol>
| |
| − | <li> Take out the tubulin monomer solution after precision purification from -80℃, and melt on the ice.
| |
| − | </li>
| |
| − | <li>Prepare 10 um tubulin monomer solution with the same concentration, and add isopyknic
| |
| − | polymerization buffer (the formula is the same as the polymerization buffer used in precision
| |
| − | purification). This step finished on the ice in order to avoid the influence of the
| |
| − | Spontaneous tubulin polymerization because of the high room temperature.
| |
| − | </li>
| |
| − | <li> Prepare a series concentration gradient of taxol solution and add the
| |
| − | tubulin polymerization system.
| |
| − | </li>
| |
| − | <li> Incubate for 1h at 37℃.</li>
| |
| − | <li> Use Nanodrop UV-IS to determinate absorbance value, and set measurement parameters 280 nm, 350
| |
| − | nm and 750 nm, but we only read at 350 nm.
| |
| − | </li>
| |
| − | <li> Use isopyknic mixed solution of polymerization buffer and PEM buffer to calibrate baseline,
| |
| − | each group of samples detect three times in parallel.
| |
| − | </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h2>Improvement</h2>
| |
| − | <h3>Culture and collection</h3>
| |
| − | <ol>
| |
| − | <li>Use LB medium to preculture transformed media 5 ml for 12h, 200 rpm/ 37℃.</li>
| |
| − | <li>
| |
| − | Culture 5 mL preculture media into 100 mL TB medium for about 3h until OD<sub>600</sub>=0.4～0.6, 200 rpm/
| |
| − | 37℃.
| |
| − | </li>
| |
| − | <li>Add arabinose or turn to 42℃ to induce P(3HB) expression for 72h, 220 rpm.</li>
| |
| − | <li>Collect cells and centrifuge for 3min, 5,000 rpm.</li>
| |
| − | <li>Remove supernatant and suspend with pure water.</li>
| |
| − | <li>Centrifuge again for 3min, 5,000 rpm and remove its supernatant.</li>
| |
| − | </ol>
| |
| − |
| |
| − |
| |
| − | <h3>Extract PHB<sup>1</sup></h3>
| |
| − | <ul>
| |
| − | <li>Centrifuge settings: 4000RPM, 10mins.</li>
| |
| − | <li> Scale as appropriate.</li>
| |
| − | <li> After each centrifuge step the supernatant should be poured off.</li>
| |
| − | </ul>
| |
| − | <ol>
| |
| − | <li>Resuspend precipitation in 10 mL Triton X-100(1% v/v in PBS) for 30mins at room temp.</li>
| |
| − | <li>Centrifuge, resuspend in 10 mL PBS.</li>
| |
| − | <li>Centrifuge, add 10 mL sodium hyperchlorite solution and incubate at 30˚C for 1 hour.</li>
| |
| − | <li>Centrifuge, wash with 10 mL 70% EtOH.</li>
| |
| − | <li>Allow powder to dry.</li>
| |
| − | </ol>
| |
| − | <p style="color: #5c5c5c;font-size: 12px;">[1]: Shahryar Shakeri, Comparison of intracellular
| |
| − | polyhydroxybutyrate granules formation between different bacterial cell subpopulations by flow
| |
| − | cytometry, Jundishapur Journal of Microbiology 2011.</p>
| |
| − | <h3>Agents formula</h3>
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="7">TB medium (1 L)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">yeast extract</td>
| |
| − | <td align="center">tryptone</td>
| |
| − | <td align="center">K<sub>2</sub>HPO<sub>4</sub></td>
| |
| − | <td align="center">KH<sub>2</sub>PO<sub>4</sub></td>
| |
| − | <td align="center">Glycerol</td>
| |
| − | <td align="center">Glucose</td>
| |
| − | <td align="center">ddH<sub>2</sub>O</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">24 g</td>
| |
| − | <td align="center">12 g</td>
| |
| − | <td align="center">72 mM</td>
| |
| − | <td align="center">17 mM</td>
| |
| − | <td align="center">8 mL</td>
| |
| − | <td align="center">2%</td>
| |
| − | <td align="center">add to 1 L</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tr>
| |
| − | <th style="text-align: center" colspan="6">PBS (1 L)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">NaCl</td>
| |
| − | <td align="center">KCl</td>
| |
| − | <td align="center">Na<sub>2</sub>HPO<sub>4</sub>·12H<sub>2</sub>O</td>
| |
| − | <td align="center">KH<sub>2</sub>PO<sub>4</sub>·3H<sub>2</sub>O</td>
| |
| − | <td align="center">pH</td>
| |
| − | <td align="center">ddH<sub>2</sub>O</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">8 g</td>
| |
| − | <td align="center">0.2 g</td>
| |
| − | <td align="center">3.63 g</td>
| |
| − | <td align="center">0.31 g</td>
| |
| − | <td align="center">adjust to 7.4</td>
| |
| − | <td align="center">add to 1 L</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <br />
| |
| − |
| |
| − | <h2>Fluorescence Detection</h2>
| |
| − | <h3>Protein functional Test</h3>
| |
| − | <ol>
| |
| − | <li> Bacteria culturing and inducing for expression. </li>
| |
| − | <li> Collect supernatant after ultrasonication. </li>
| |
| − | <li>
| |
| − | Concentrate the supernatant via ultra-filtering
| |
| − | <table class="table">
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th style="text-align: center">Protein samples</th>
| |
| − | <th style="text-align: center">α-Tubulin YNE</th>
| |
| − | <th style="text-align: center">α-tubulin-YCE</th>
| |
| − | <th style="text-align: center">β-tubulin</th>
| |
| − | <th style="text-align: center">Aggregation buffer</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">75 μL</td>
| |
| − | <td align="center">75 μL</td>
| |
| − | <td align="center">150 μL</td>
| |
| − | <td align="center">300 μL</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | <table class="table">
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th style="text-align: center">Protein samples</th>
| |
| − | <th style="text-align: center">α-tubulin-YCE</th>
| |
| − | <th style="text-align: center">β-tubulin- YNE</th>
| |
| − | <th style="text-align: center">Aggregation buffer</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">150 μL</td>
| |
| − | <td align="center">150 μL</td>
| |
| − | <td align="center">300 μL</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − |
| |
| − | </li>
| |
| − | <li> Mixed the protein samples and add proper GTP, taxol to 200uM. </li>
| |
| − | <li> Use absolute recording spectrofluorometer with 514nm excitation. </li>
| |
| − | <li> Record light intensity in 520nm-530nm wavelength emission. </li>
| |
| − | </ol>
| |
| − |
| |
| − | <h3>Taxol-concentration based assay</h3>
| |
| − |
| |
| − | <ol>
| |
| − | <li> Bacteria culturing and inducing for expression </li>
| |
| − | <li> Collect supernatant after ultrasonication </li>
| |
| − | <li>
| |
| − | Concentrate the supernatant via ultra-filtering
| |
| − | <table class="table">
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <th style="text-align: center">Protein samples</th>
| |
| − | <th style="text-align: center">α-Tubulin YNE</th>
| |
| − | <th style="text-align: center">α-tubulin-YCE</th>
| |
| − | <th style="text-align: center">β-tubulin</th>
| |
| − | <th style="text-align: center">Aggregation buffer</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td align="center">Dosage</td>
| |
| − | <td align="center">75 μL</td>
| |
| − | <td align="center">75 μL</td>
| |
| − | <td align="center">150 μL</td>
| |
| − | <td align="center">300 μL</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </li>
| |
| − | <li>
| |
| − | Mixed the protein samples and add taxol respectively as the following table:
| |
| − | <table class="table">
| |
| − | <tbody>
| |
| − | <tr>
| |
| − | <td align="center">Taxol concentration (μM)</td>
| |
| − | <td align="center">0</td>
| |
| − | <td align="center">0.5</td>
| |
| − | <td align="center">5</td>
| |
| − | <td align="center">25</td>
| |
| − | <td align="center">50</td>
| |
| − | <td align="center">100</td>
| |
| − | <td align="center">250</td>
| |
| − | <td align="center">500</td>
| |
| − | </tr>
| |
| − | </tbody>
| |
| − | </table>
| |
| − | </li>
| |
| − | <li> Use absolute recording spectrofluorometer with 514nm excitation </li>
| |
| − | <li> Record light intensity in 520nm-530nm wavelength emission. </li>
| |
| − | </ol>
| |
| | | | |
| | </article> | | </article> |
