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<li><a href="#section2">Reaction System<br> of Colony PCR</a></li> | <li><a href="#section2">Reaction System<br> of Colony PCR</a></li> | ||
<li><a href="#section3">SDS-PAGE</a></li> | <li><a href="#section3">SDS-PAGE</a></li> | ||
+ | <li><a href="#section3">Titration</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
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<h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Construction of the whole circuit</h3> | ||
<p><strong>The protocol of SDS-PAGE:</strong><p> | <p><strong>The protocol of SDS-PAGE:</strong><p> | ||
− | <p> | + | <p>1. Take 1ml of the bacterial suspension(OD600 is about 2.0), add 9ml LB medium, culture the bacteria in a shaker until its OD600 is 0.6. </p> |
− | <p> | + | <p>2. Add 1 ‰ IPTG to induce our engineering bacteria, and culture them for 5 hours.</p> |
− | <p> | + | <p>3. Centrifuge the bacterial suspension, and gently rinse it, add utrapure water into the cultures until its OD600 is about 2.0.</p> |
− | <p> | + | <p>4. Prepare different concentrations of terbium chloride solution of 40ml. Adding 3ml bacteria suspension to each of them respectively.</p> |
− | <p> | + | <p>5. Add excess sodium hydroxide to the supernatant until all the ions are precipitated. Centrifugate.</p> |
+ | <p>6. The resulting precipitate was titrated with hydrogen chloride to give the titration results.</p> | ||
</div> | </div> | ||
+ | <div id="section4" style="border: solid 1px #666; margin:5px;border-radius:10px;overflow: hidden;"> | ||
+ | <h3 style="background-color:#ce6b9a;color:#ffffff; padding:10px;letter-spacing:1px; margin-top:0;">Titration</h3> | ||
+ | <p>We transferred the plasmids carrying the sequences encoding 3×LBT into BL21, and set a control group in which 3×LBT is not involved in the plasmids transferred. Both can be expressed with the induction of IPTG. Having cultivated the bacteria into similar densities, we add IPTG and then incubate them for 5 hours. Afterwards, we boiled the bacteria to release the proteins on their cell membrane. Then we detected the proteins we extracted by SDS-PAGE. As a result, bacteria in the test group expressed a large quantity of LBT while those in the control group did not. | ||
+ | </p> | ||
+ | |||
+ | |||
</div> | </div> | ||
+ | |||
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Revision as of 18:26, 1 November 2017