Difference between revisions of "Team:Imperial College/Protocols"

 
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                 <div class="titleimg">
 
                 <div class="titleimg">
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                         <h1>Protocols</h1>     
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                         <h1>References</h1>     
 
                         <br/>
 
                         <br/>
 
                         <br/>
 
                         <br/>
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               </div>
 
               </div>
  
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          <h3>Reason for sharing this:</h3>
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<p>Anderson, A., Laohavisit, A., Blaby, I., Bombelli, P., Howe, C., Merchant, S., Davies, J. and Smith, A. (2015). Exploiting algal NADPH oxidase for biophotovoltaic energy. Plant Biotechnology Journal, 14(1), pp.22-28.
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            <h3>Workflow</h3>
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Brigelius-Flohé, R. and Flohé, L. (2011). Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors. Antioxidants & Redox Signaling, 15(8), pp.2335-2381.
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            <h3>Recipes</h3>
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Buldum, G., Bismarck, A. and Mantalaris, A. (2017). Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Bioprocess and Biosystems Engineering, 41(2), pp.265-279.
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Esvelt, K., Carlson, J. and Liu, D. (2011). A system for the continuous directed evolution of biomolecules. Nature, 472(7344), pp.499-503.
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        <div class="accordion">
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Holmes, D., Walker, D., Dang, Y. and Lovley, D. (2016). The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microbial Genomics, 2(8).
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        <button class="collapsible">TSS Chemically Competent Cell Preparation</button>       
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Hsu, T., Welner, D., Russ, Z., Cervantes, B., Prathuri, R., Adams, P. and Dueber, J. (2018). Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature Chemical Biology, 14(3), pp.256-261.
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        <div class="drop">
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Kim, H., Jeong, H., Hwang, S., Lee, M., Lee, Y., Lee, D. and Lee, S. (2014). Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase. Frontiers in Microbiology, 5.
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Kraj, A., Brouwer, H., Reinhoud, N. and Chervet, J. (2013). A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection. Analytical and Bioanalytical Chemistry, 405(29), pp.9311-9320.
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                <p>Materials</p>
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Robinson, R. (2013). A Little Switch: Alternative Domain Conformations Control Bacterial Flagella Rotation Direction. PLoS Biology, 11(2), p.e1001480.
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Shadrin, A., Sheppard, C., Severinov, K., Matthews, S. and Wigneshweraraj, S. (2012). Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised. Microbiology, 158(Pt_11), pp.2753-2764.
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                <ul>
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Sheplock, R., Recinos, D., Mackow, N., Dietrich, L. and Chander, M. (2012). Species-specific residues calibrate SoxR sensitivity to redox-active molecules. Molecular Microbiology, 87(2), pp.368-381.
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                    <li>LB media
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Wei, T., Cheng, B. and Liu, J. (2016). Genome engineering Escherichia coli for L-DOPA overproduction from glucose. Scientific Reports, 6(1).<p>
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                        <ul>
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                            <li>1% w/v Peptone</li>
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                            <li>1% w/v NaCl</li>
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                            <li>0.5% w/v Yeast Extract</li>
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                        </ul>
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                    </li>
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                    <li>TSS Solution
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                        <ul>
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                            <li>85 ml sterile LB media</li>
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                            <li>10 g PEG-3350 in 5 ml H<sub>2</sub>O (10%) </li>
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                            <li>5 ml DMSO (5%) </li>
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                            <li>2 ml of 1M MgCl<sub>2</sub> (Hydrous: 1.017g + 5ml H20. Anhydrous: 0.476g + 5mL H2O)</li>
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                        </ul>
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                    </li>
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                    <li>KCM Solution
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                        <ul>
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                            <li>500 ml H<sub>2</sub>O </li>
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                            <li>18.64 g KCl (500mM)</li>
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                            <li>8.32 g CaCl<sub>2</sub> (150mM)</li>
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                        </ul>
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                    </li>
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                    <li>Two 2 L conical flasks </li>
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                    <li>Four 250ml centrifuge bottles </li>
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                    <li>High-speed centrifuge </li>
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                </ul>
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                <p >Procedure</p>
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                <ul>
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                    <li >Day 1
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                        <ol>
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                            <li>Prepare two 500 ml aliquots of LB media in two 2 L conical flasks and autoclave</li>
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                            <li>Autoclave four 250 ml centrifuge bottles</li>
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                            <li>From -80ᵒC stocks, streak plate cells to be made competent on agar plates supplemented with appropriate antibiotics. Note: It is advised best not to re-streak from a competent cell stock.</li>
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                        </ol>
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                    </li>
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                    <li>Day 2
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                        <ol>
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                            <li>At the end of the day, pick tree colonies to be grown overnight in three separate aliquots of 10 ml of LB media supplemented with the appropriate antibiotics.</li>
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                            <li>Incubate the cells overnight at 37ᵒC, in a shaking incubator set to 210 RPM.</li>
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                        </ol>
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                    </li>
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                    <li >Day 3
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                        <ol>
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                            <li>In the morning inoculate the 500 ml flasks of LB media with 5 ml of the overnight cell culture. </li>
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                            <li>Incubate the cells at 37ᵒC, in a shaking incubator set to 210 RPM. Measure the OD600 every hour until OD600 0.4 - 0.6 is achieved.</li>
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                            <li>While the cells incubate, chill the following:
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                                <ul>
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                                    <li>The four 250 ml centrifuge bottles – on ice</li>
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                                    <li>Labelled PCR tubes (prepare 300 for a 1 L cell culture) – on ice</li>
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                                    <li>Set the centrifuge to 4ᵒC and leave the rotor inside</li>
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                                    <li>50 ml TSS solution </li>
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                                </ul>
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                            </li>
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                            <li>Once a OD600 of 0.4-0.6 is achieved, aliquot the culture into the centrifuge tubes and keep on ice to inhibit further growth.</li>
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                            <li>Pellet the cells in the centrifuge by spinning them at 5000 g for 5 min</li>
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                            <li>Discard the supernatant and resuspend the cells in 50 ml ice-cold TSS</li>
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                            <li>Aliquot 200 µl of cell solution into each PCR tube</li>
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                            <li>Directly store the tubes in a -80ᵒC freezer. </li>
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                        </ol>
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                    </li>
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                </ul>
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                <p >Source:</p>
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                <p1>Chung, C., Niemela, S. and Miller, R. (1989). One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. <i>Proceedings of the National Academy of Sciences, 86(7)</i>, pp.2172-2175.</p1>                       
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        </div>
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    </div>   
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        <div class="accordion">
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            <button class="collapsible">Miniprep Kit Protocol</button>       
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        <div class="drop">
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                <p>Materials</p>
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            <ul>
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                <li>Lorem ipsum dolor sit amet consectetur adipisicing elit. Obcaecati rerum magni, libero ut fuga doloribus impedit dolorem laudantium, sunt error vitae repellat quia, quidem nihil accusamus. Omnis unde quasi cum.</li>
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                <table class="protocolTable">
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                    <tr>
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                        <th>Sample</th>
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                        <th>Reagent 1</th>
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                        <th>Reagent 2</th>
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                        <th>Water</th>
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                        <th>Total volume</th>
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                    </tr>
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                    <tr>
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                        <td>No.1</td>
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                        <td>3</td>
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                        <td>3</td>
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                        <td>4</td>
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                        <td>10</td>
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                    </tr>
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                    <tr>
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                        <td>No.2</td>
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                        <td>4</td>
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                        <td>3</td>
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                        <td>3</td>
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                        <td>10</td>
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                    </tr>
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                    <tr>
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                        <td>No.3</td>
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                        <td>5</td>
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                        <td>3</td>
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                        <td>2</td>
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                        <td>10</td>
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                    </tr>
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                </table>
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                <li>Lorem ipsum dolor sit amet consectetur adipisicing elit. Obcaecati rerum magni, libero ut fuga doloribus impedit dolorem laudantium, sunt error vitae repellat quia, quidem nihil accusamus. Omnis unde quasi cum.</li>
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            </ul> 
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            <p >Procedure</p> 
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            <p >Source:</p>           
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        </div>
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<div class="accordion">
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        <button class="collapsible">Making Agar Plates</button>       
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        <div class="drop">
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                  <p>Materials</p>
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            <ul>
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                <li>Lorem ipsum dolor sit amet consectetur adipisicing elit. Obcaecati rerum magni, libero ut fuga doloribus impedit dolorem laudantium, sunt error vitae repellat quia, quidem nihil accusamus. Omnis unde quasi cum.</li>
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                <li>Lorem ipsum dolor sit amet consectetur adipisicing elit. Obcaecati rerum magni, libero ut fuga doloribus impedit dolorem laudantium, sunt error vitae repellat quia, quidem nihil accusamus. Omnis unde quasi cum.</li>
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            </ul> 
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            <p >Procedure</p> 
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            <p >Source:</p>         
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                </div> 
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  <script>
 
  <script>

Latest revision as of 02:56, 18 October 2018


References



Anderson, A., Laohavisit, A., Blaby, I., Bombelli, P., Howe, C., Merchant, S., Davies, J. and Smith, A. (2015). Exploiting algal NADPH oxidase for biophotovoltaic energy. Plant Biotechnology Journal, 14(1), pp.22-28. Brigelius-Flohé, R. and Flohé, L. (2011). Basic Principles and Emerging Concepts in the Redox Control of Transcription Factors. Antioxidants & Redox Signaling, 15(8), pp.2335-2381. Buldum, G., Bismarck, A. and Mantalaris, A. (2017). Recombinant biosynthesis of bacterial cellulose in genetically modified Escherichia coli. Bioprocess and Biosystems Engineering, 41(2), pp.265-279. Esvelt, K., Carlson, J. and Liu, D. (2011). A system for the continuous directed evolution of biomolecules. Nature, 472(7344), pp.499-503. Holmes, D., Walker, D., Dang, Y. and Lovley, D. (2016). The electrically conductive pili of Geobacter species are a recently evolved feature for extracellular electron transfer. Microbial Genomics, 2(8). Hsu, T., Welner, D., Russ, Z., Cervantes, B., Prathuri, R., Adams, P. and Dueber, J. (2018). Employing a biochemical protecting group for a sustainable indigo dyeing strategy. Nature Chemical Biology, 14(3), pp.256-261. Kim, H., Jeong, H., Hwang, S., Lee, M., Lee, Y., Lee, D. and Lee, S. (2014). Short-term differential adaptation to anaerobic stress via genomic mutations by Escherichia coli strains K-12 and B lacking alcohol dehydrogenase. Frontiers in Microbiology, 5. Kraj, A., Brouwer, H., Reinhoud, N. and Chervet, J. (2013). A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection. Analytical and Bioanalytical Chemistry, 405(29), pp.9311-9320. Robinson, R. (2013). A Little Switch: Alternative Domain Conformations Control Bacterial Flagella Rotation Direction. PLoS Biology, 11(2), p.e1001480. Shadrin, A., Sheppard, C., Severinov, K., Matthews, S. and Wigneshweraraj, S. (2012). Substitutions in the Escherichia coli RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2 and RNA polymerase becomes compromised. Microbiology, 158(Pt_11), pp.2753-2764. Sheplock, R., Recinos, D., Mackow, N., Dietrich, L. and Chander, M. (2012). Species-specific residues calibrate SoxR sensitivity to redox-active molecules. Molecular Microbiology, 87(2), pp.368-381. Wei, T., Cheng, B. and Liu, J. (2016). Genome engineering Escherichia coli for L-DOPA overproduction from glucose. Scientific Reports, 6(1).