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<li><a href="#section-9">Point mutation</a></li> | <li><a href="#section-9">Point mutation</a></li> | ||
<li><a href="#section-10">Reagents</a></li> | <li><a href="#section-10">Reagents</a></li> | ||
− | <li><a href="#section-11"> | + | <li><a href="#section-11">Whole cell catalysis</a></li> |
<li><a href="#section-12">the LiAc SS carrier DNA PEG method</a></li> | <li><a href="#section-12">the LiAc SS carrier DNA PEG method</a></li> | ||
− | <li><a href="#section-13"> | + | <li><a href="#section-13">References</a></li> |
</ul> | </ul> | ||
</div> | </div> | ||
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</h4> | </h4> | ||
− | <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;"> | + | <h2 id="section-11" style="padding-top: 100px; margin-top: -50px;">Whole-cell catalysis</h2> |
− | <h4> | + | <h4> |
− | <br /> | + | 1. Prepare sterile tubes of 5 ml of 2YT+antibiotics. Use a sterile pipet tip to pick bacteria from plates. Throw the tip |
− | <br /> | + | into the tubes. Incubate in a rotary shaker at37℃ for 3-4h.<br /> 2. Transfer 120µl of bacteria from |
− | <br /> | + | a slant culture into an Erlenmeyer flask containing 60 mL LB medium with appropriate resistance, incubate at |
− | <br /> | + | 37 °C. |
− | <br /> | + | <br /> 3. When the OD600 reaches 0.6-0.8, the induction of IPTG (0.5 mM) should be carried out. Incubate at 30 |
+ | °C on a rotary shaker incubator at 220 rpm for 14 h.<br /> 4. Harvest the bacteria(6000rpm/min 7min). Wash with | ||
+ | 30ml PBS.<br /> 5. The biocatalytic reaction mixture contained 10% glycerol, E▪ coli and 50mM PBS. Reaction time | ||
+ | gradient: 8h, 16h, 32h.<br /> 6. Use HPLC for further analysis.<br /> | ||
+ | <br /> Reference: Li N, He Y, Chen Y, et al. Production of cyclic adenosine-3′,5′-monophosphate by whole cell | ||
+ | catalysis using recombinant Escherichia coli, overexpressing adenylate cyclase[J]. Korean Journal of Chemical | ||
+ | Engineering, 2013, 30(4):913-917.<br /> | ||
</h4> | </h4> | ||
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</h4> | </h4> | ||
− | <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;"> | + | <h2 id="section-13" style="padding-top: 100px; margin-top: -50px;">References</h2> |
<h4> | <h4> | ||
− | + | http://parts.igem.org/Help:Protocols/Transformation<br /> https://2015.igem.org/Team:Aachen/Project/Overview | |
− | + | <br /> http://www.zymoresearch.com/category/all-products | |
− | + | <br /> http://www.corning.com/worldwide/en/products/life-sciences/resources/brands/axygen-brand-products.html | |
− | + | <br /> http://www.tsingke.net/shop/ | |
− | <br /> | + | <br /> http://www.cwbiotech.com/ |
− | + | <br /> | |
− | + | ||
− | + | ||
− | <br /> | + | |
− | + | ||
− | + | ||
</h4> | </h4> | ||
Revision as of 14:43, 30 October 2017