Difference between revisions of "Team:Munich/Improve"
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<td id="myContent" width="20%" valign=top align=center> | <td id="myContent" width="20%" valign=top align=center> | ||
| + | <a href="#-"> | ||
| + | <div class="popup" id="OH_PCR_Experiment_Popup"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Munich--Improve_TEV_OH_PCR.png"> | ||
| + | </div></a> | ||
| + | <a href="#-"> | ||
| + | <div class="popup" id="TEV_SEC_Popup"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/f/f1/T--Munich--Improve_TEV_SEC_SDS.png"> | ||
| + | </div></a> | ||
| + | <a href="#-"> | ||
| + | <div class="popup" id="TEV_Activity_Popup"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/6/6b/T--Munich--Improve_TEV_Cleavage_final.png"> | ||
| + | </div></a> | ||
<br> | <br> | ||
<!-- Head End --> | <!-- Head End --> | ||
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</p> | </p> | ||
<p> | <p> | ||
| − | For scientists the TEV protease is a molecular tool to cleave of all sorts of protein tags precisely due to its sequence specificity. It recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Gln-Ser and cleaves then between glutamic acid and serine. In our project, the TEV protease is a main component in the Intein-Extein readout, but also was used in the purification procedure of our Cas13a proteins. We improved the BioBrick <a class="myLink" href="http://parts.igem.org/Part:BBa_K1319008">BBa_K1319008</a> by adding a His<sub>6</sub>-tag, which made it possible to purify this protease. | + | For scientists the TEV protease is a molecular tool to cleave of all sorts of protein tags precisely due to its sequence specificity. It recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Gln-Ser and cleaves then between glutamic acid and serine. In our project, the TEV protease is a main component in the Intein-Extein readout, but also was used in the purification procedure of our Cas13a proteins. <b>We improved the BioBrick <a class="myLink" href="http://parts.igem.org/Part:BBa_K1319008">BBa_K1319008</a> by adding a His<sub>6</sub>-tag, which made it possible to purify this protease. </b> We show here the characterization of our improved BioBrick, but the completed details are available in the Registry page: <a class="myLink" href="http://parts.igem.org/Part:BBa_K2323002">BBa_K2323002</a>. |
</p> | </p> | ||
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<div class="captionPicture"> | <div class="captionPicture"> | ||
<img height=400 src="https://static.igem.org/mediawiki/2017/3/36/T--Munich--Improve_Plasmid_Map.svg"> | <img height=400 src="https://static.igem.org/mediawiki/2017/3/36/T--Munich--Improve_Plasmid_Map.svg"> | ||
| − | <p> | + | <p><b>Figure 1:</b>The TEV plasmid map shows the binding sites of the overhang primers. Indicated are also coding sequence, terminator, T7 promotor and RBS</p> |
| − | The TEV plasmid map shows the binding sites of the overhang primers. Indicated are also coding sequence, terminator, T7 promotor and RBS | + | |
| − | </p> | + | |
</div> | </div> | ||
</td> | </td> | ||
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After PCR we ligated the plasmid using the T4 ligase. This sample was then transformed in <i>E. coli</i> DH5α for plasmid storage and <i>E. coli</i> BL21star for protein expression. We expressed the TEV protease in 2xYT medium and purified it via <a class="myLink" href="/Team:Munich/Protocols">affinity and size exclusion chromatography</a>. | After PCR we ligated the plasmid using the T4 ligase. This sample was then transformed in <i>E. coli</i> DH5α for plasmid storage and <i>E. coli</i> BL21star for protein expression. We expressed the TEV protease in 2xYT medium and purified it via <a class="myLink" href="/Team:Munich/Protocols">affinity and size exclusion chromatography</a>. | ||
</p> | </p> | ||
| + | <div class="captionPicture"> | ||
| + | <a href="#TEV_SEC_Popup"><img height=300 src="https://static.igem.org/mediawiki/2017/f/f1/T--Munich--Improve_TEV_SEC_SDS.png"></a> | ||
| + | <p><b>Figure 2:</b> The TEV plasmid map shows the binding sites of the overhang primers. Indicated are also coding sequence, terminator, T7 promotor and RBS</p> | ||
| + | </div> | ||
</td> | </td> | ||
<td colspan=3 align=center valign=center> | <td colspan=3 align=center valign=center> | ||
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<div class="captionPicture"> | <div class="captionPicture"> | ||
<a href="#OH_PCR_Experiment_Popup"><img width=300 src="https://static.igem.org/mediawiki/2017/1/1b/T--Munich--Improve_TEV_OH_PCR.png"></a> | <a href="#OH_PCR_Experiment_Popup"><img width=300 src="https://static.igem.org/mediawiki/2017/1/1b/T--Munich--Improve_TEV_OH_PCR.png"></a> | ||
| − | <p><b>Figure | + | <p><b>Figure 3:</b> PCR overhang for TEV His-tag</p> |
</div> | </div> | ||
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</tr> | </tr> | ||
| − | <tr><td align=center valign=center colspan= | + | <tr><td align=center valign=center colspan=6> |
<div class="captionPicture"> | <div class="captionPicture"> | ||
| − | <img height= | + | <img height=600 src="https://static.igem.org/mediawiki/2017/c/c1/T--Munich--Improve_TEV_SEC.svg"> |
| − | <p><b>Figure | + | <p><b>Figure 4:</b> The TEV plasmid map shows the binding sites of the overhang primers. Indicated are also coding sequence, terminator, T7 promotor and RBS</p> |
| − | + | ||
| − | + | ||
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| − | + | ||
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</div> | </div> | ||
</td> | </td> | ||
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<tr><td align=center valign=center colspan=6> | <tr><td align=center valign=center colspan=6> | ||
| − | <p> | + | <p> |
For an activity test, we incubated 30 µg His-MBP-Cas13a-Lsh as substrate with 1 µg of our TEV protease. We inactivated the cleavage reaction by adding 1x SDS-loading buffer. We analyzed the reaction with a SDS-PAGE and loaded samples, which were incubated 0, 1, 2, 3, 4 ,5 and overnight. The gel shows that nearly all our substrate is already cleaved after 1 h into His-MBP and Cas13a-Lsh. | For an activity test, we incubated 30 µg His-MBP-Cas13a-Lsh as substrate with 1 µg of our TEV protease. We inactivated the cleavage reaction by adding 1x SDS-loading buffer. We analyzed the reaction with a SDS-PAGE and loaded samples, which were incubated 0, 1, 2, 3, 4 ,5 and overnight. The gel shows that nearly all our substrate is already cleaved after 1 h into His-MBP and Cas13a-Lsh. | ||
</p> | </p> | ||
| − | <img width=600 src="https://static.igem.org/mediawiki/2017/6/6b/T--Munich--Improve_TEV_Cleavage_final.png"> | + | <div class="captionPicture"> |
| − | <p> | + | <a href="#TEV_Activity_Popup"><img width=600 src="https://static.igem.org/mediawiki/2017/6/6b/T--Munich--Improve_TEV_Cleavage_final.png"></a> |
| − | Next, the activity should be analyzed between 0 and 1 h to correctly evaluate the results. However, we highly purified our His-TEV protease and also used it successfully to process our Cas13a proteins. Here, we provide a BioBrick, which could be useful for all future iGEM teams.</p | + | <p><b> Figure 5:</b> The SDS-PAGE showing the cleavage of our substrate after respective incubation time</p> |
| − | + | </div> | |
| + | <p>Next, the activity should be analyzed between 0 and 1 h to correctly evaluate the results. However, we highly purified our His-TEV protease and also used it successfully to process our Cas13a proteins. Here, we provide a BioBrick, which could be useful for all future iGEM teams.</p> | ||
</tr> | </tr> | ||
Latest revision as of 03:26, 2 November 2017
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