Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602040"

 
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<h3> K1602040 - B0034-enhanced yellow fluorescent protein-GBD-ligand (B0034-eYFP-GBDlig)</h3>
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<h3> K1602040 - T7-B0034-enhanced yellow fluorescent protein-GBD-ligand (T7-B0034-eYFP-GBDlig)</h3>
  
  
 
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<img src="https://static.igem.org/mediawiki/2015/5/59/Bild_1%3BColony_PCR_of_B0034-YFP.png" width=100% height=100%>
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<figcaption><br><b>Figure 1</b> Colony PCR of B0034-YFP (1). Colony 1 contained the insert of around 1.1 kbp. DNA marker: Quick-Load Purple 2-Log DNA Ladder.</figcaption>
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The gene <em>YFP</em> encoding for the yellow fluorescent protein was provided by the iGEM Registry. Competent E. coli cells of strain Top 10 were transformed via heat shock with the vector pSB1C3 harboring the coding sequence. Colonies were inoculated and the plasmids were extracted. The RBS was added by cloning the construct into the vector pSB1A2 containing the RBS sequence. Therefor, the construct was digested with <em>Xba</em>I and <em>Pst</em>I and ligated into the vector, which was digested with <em>Spe</em>I and <em>Pst</em>I. Competent <em>E. coli </em> Top 10 were transformed via heat shock. Positive colonies were determined by colony PCR with VF2 and VR and inoculated, the plasmids were extracted. To connect the enzyme to the <em>in vitro</em> scaffold a linker was added. Therefor, the construct B0034-YFP_fus_rev was used as template for a PCR with VF2 and GBDlig_opt_rev. 1 ng of the purified PCR product was used for the next PCR with VF2 and GBDlig_opt_rev2.
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<a href=" https://2015.igem.org/Team:TU_Darmstadt/Notebook/sec1/K1602037 " title="Opens internal link in current window" class="internal link">K1602037</a> was digested with <em>Xba</em>I and <em>Pst</em>I and ligated into T7-pSB1A2. Competent <em>E. coli</em> Top 10 were transformed via heat shock. The plasmids were extracted and digested with <em>EcoR</em>I and <em>Pst</em>I and cloned into pSB1C3. Competent <em>E. coli</em> Top 10 were transformed and colonies were screened by colony PCR. Plasmids were extracted and used for protein expression in <em>E. coli</em> strain BL21. </p>
 
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Latest revision as of 07:38, 18 September 2015

K1602040 - T7-B0034-enhanced yellow fluorescent protein-GBD-ligand (T7-B0034-eYFP-GBDlig)


K1602037 was digested with XbaI and PstI and ligated into T7-pSB1A2. Competent E. coli Top 10 were transformed via heat shock. The plasmids were extracted and digested with EcoRI and PstI and cloned into pSB1C3. Competent E. coli Top 10 were transformed and colonies were screened by colony PCR. Plasmids were extracted and used for protein expression in E. coli strain BL21.