Difference between revisions of "Team:TU Darmstadt/Notebook/sec1/K1602001"

 
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<h3>K1602001 - D-xylonolactonase with strong RBS (B0034-xylC)</h3>
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<h3>K1602001 - B0034-<small>D</small>-Xylonolactonase (B0034-xylC)</h3>
  
  
 
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<img class="transparent" alt="https://static.igem.org/mediawiki/2015/c/c3/TU_Darmstadt_Gel_B0034-xylB%2CB0034-xylC%2CB0034-yagE.png" src="https://static.igem.org/mediawiki/2015/c/c3/TU_Darmstadt_Gel_B0034-xylB%2CB0034-xylC%2CB0034-yagE.png">
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<figcaption><br><b>Figure 1</b> PCR of cadA (X). The size of the amplified product was around 1.5 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
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<figcaption><br><b>Figure 1</b> Gel of B0034-<i>xylC</i>. The size of the amplified product was around 1 kbp. DNA marker: 2-Log DNA Ladder (NEB).</figcaption>
 
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<p>The BBa_B0034 ribosomal binding site was added upstream of the <em>xylC</em>-gene by cloning said gene in the <em>pSB1A2‑</em>vector containing the RBS.</p>
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<p>The BBa_B0034 ribosomal binding site was added upstream of the <a href=" https://2015.igem.org/Team:TU_Darmstadt/Notebook/sec1/K1602010 " title="Opens internal link in current window" class="internal link">xylC</a>-gene by cloning the gene in the pSB1A2-vector containing the RBS.</p>
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<p>The <em>pSB1A2‑</em>vector was cut using <em>Spe</em>I<em>‑</em> and <em>Pst</em>I‑restriction enzymes and later dephosphorylated. The gene was digested using <em>Xba</em>I and <em>Spe</em>I and ligated in the vector with <em>T4</em>‑ligase. After transformation in <em>E. coli </em>Top10 the resulting colonies were screened for positive clones by performing colony‑PCRs with the VF2 and VR standard-oligonucleotides. The plasmids of positive clones were purified and the inserts verified by sanger-sequencing. Afterwards the <em>pSB1A2‑</em>vector was replaced by the <em>pSB1C3</em> registry standard vector using <em>EcoR</em>I and <em>Spe</em>I. The dephosphorylation and ligation was performed.</p>
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<p>The pSB1A2‑vector was digested using <em>Spe</em>I and <em>Pst</em>I and later dephosphorylated. The gene was digested using <em>Xba</em>I and <em>Spe</em>I and ligated in the vector with T4‑ligase. After transformation of <em>E. coli </em>Top 10 the resulting colonies were screened for positive clones by performing colony PCRs with VF2 and VR oligonucleotides. The plasmids of positive clones were purified and the inserts verified by sanger-sequencing. Afterwards the pSB1A2‑vector was replaced by the pSB1C3 vector using <em>EcoR</em>I and <em>Spe</em>I. The dephosphorylation and ligation were performed.</p>
 
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Latest revision as of 23:34, 18 September 2015

K1602001 - B0034-D-Xylonolactonase (B0034-xylC)


https://static.igem.org/mediawiki/2015/c/c3/TU_Darmstadt_Gel_B0034-xylB%2CB0034-xylC%2CB0034-yagE.png

Figure 1 Gel of B0034-xylC. The size of the amplified product was around 1 kbp. DNA marker: 2-Log DNA Ladder (NEB).

The BBa_B0034 ribosomal binding site was added upstream of the xylC-gene by cloning the gene in the pSB1A2-vector containing the RBS.

The pSB1A2‑vector was digested using SpeI and PstI and later dephosphorylated. The gene was digested using XbaI and SpeI and ligated in the vector with T4‑ligase. After transformation of E. coli Top 10 the resulting colonies were screened for positive clones by performing colony PCRs with VF2 and VR oligonucleotides. The plasmids of positive clones were purified and the inserts verified by sanger-sequencing. Afterwards the pSB1A2‑vector was replaced by the pSB1C3 vector using EcoRI and SpeI. The dephosphorylation and ligation were performed.