Team:Tuebingen/Project/Plasmids

From 2014.igem.org

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Since a critical part of our project was the overexpression, chosing the correct vector was crucial. Of particular importance are of course the quality of the promotor and the RBS, as well as a His-Tag for purification. All these parts are available in the parts-registry, but even with the convenient cloning of BioBricks, we thought a more conventional approach would suit our needs better. We chose a pET-vector, which we could easily modify by ligating it with annealed oligo nucleotides. The vector pETBlue-1 already came equipped with a T7-promotor and a LacZ site (see figure).<br>
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Since a critical part of our project was the overexpression, chosing the correct vector was crucial. Of particular importance are of course the quality of the promotor and the RBS, as well as a His-Tag for purification. All these parts are available in the parts-registry, but even with the convenient cloning of BioBricks, we thought a more conventional approach would suit our needs better. We chose a pET-vector, which we could easily modify by ligating it with annealed oligo nucleotides. The vector pETBlue-1 already came equipped with a T7-promotor and a LacZ site (see figure 1).<br>
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Using the oligo nucleotides depicted in figure X, we were able to create a convenient vector for IPTG-inducible overexpression of genes in the RFC25 standard. The aligned oligo nucleotides had sticky ends to ligate with XbaI and EcoRI sites, creating a scar.
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<img src="https://static.igem.org/mediawiki/2014/3/34/Tue2014_PETBlue-1_map.png" style="width: 50%">
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<p id="picTextCenter">Figure 1: Plasmid map of pETBlue-1 <a href="#Ref">[1]</a> . <i>XbaI</i> and <i>XmaI</i> were used to insert the annealed oligo nucleotides to create an easily accessible vector for overexpression.</p>
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<img src="https://static.igem.org/mediawiki/2014/4/4c/Tue2014_annealed_pET_oligos.JPG" style="width: 100%">
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<p id="picTextCenter">Figure 2: Product of olio nucleotide annealing for insertion in pETBlue-1.</p>
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Using the oligo nucleotides depicted in figure 2, we were able to create a convenient vector for IPTG-inducible overexpression of genes in the RFC25 standard. The aligned oligo nucleotides had sticky ends to ligate with XbaI and EcoRI sites, creating a scar.
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The DNA fragment from annealed oligos was to introduce:
The DNA fragment from annealed oligos was to introduce:
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The last point proved to be somewhat tricky. Unfortunately, as figure XX shows, pETBlue-1 is not optimal for RFC25 cloning, since it has both NgoMIV and AgeI restriction sites. We got around this problem by using a third enzyme with the same sticky ends: XmaI. This was to be the up-stream site for insertion.  
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The last point proved to be somewhat tricky. Unfortunately, as figure 1 shows, pETBlue-1 is not optimal for RFC25 cloning, since it has both NgoMIV and AgeI restriction sites. We got around this problem by using a third enzyme with the same sticky ends: XmaI. This was to be the up-stream site for insertion.  
Further modifications of the pET-vector were supposed to include the deletion of all NgoMIV and AgeI sites, as well as the changing of the introduced XmaI site to AgeI, so that the vector could be used with conventional RFC25 enzymes.<br>
Further modifications of the pET-vector were supposed to include the deletion of all NgoMIV and AgeI sites, as well as the changing of the introduced XmaI site to AgeI, so that the vector could be used with conventional RFC25 enzymes.<br>
The AgeI site was successfully changed using site directed mutagenesis PCR. However, the difficulty we had with expression using the modified pETBlue1.2 led us to abandon this side project.
The AgeI site was successfully changed using site directed mutagenesis PCR. However, the difficulty we had with expression using the modified pETBlue1.2 led us to abandon this side project.
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<a name="pSBX"></a>
As part of our collaboration with <a href="https://2014.igem.org/Team:Heidelberg">Team Heidelberg</a> we received two expression vectors they designed, <a href="http://parts.igem.org/Part:BBa_K1362093">pSBX1K3</a> and <a href="http://parts.igem.org/Part:BBa_K1362097">pSBX4K5</a>. We have used pSBX1K3 for test expressions of our constructs.
As part of our collaboration with <a href="https://2014.igem.org/Team:Heidelberg">Team Heidelberg</a> we received two expression vectors they designed, <a href="http://parts.igem.org/Part:BBa_K1362093">pSBX1K3</a> and <a href="http://parts.igem.org/Part:BBa_K1362097">pSBX4K5</a>. We have used pSBX1K3 for test expressions of our constructs.
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    <td><img src="https://static.igem.org/mediawiki/2014/d/d1/Tue2014_pSB1K3_Map2.jpg" style="width: 100%"></td>
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    <td></td>
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    <td><img src="https://static.igem.org/mediawiki/2014/4/47/Tue2014_pSBX4K5_Map.png" style="width: 100%"></td>
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    <td><p id="picTextCenter">Figure 3: Plasmid map of pSBX1K3; high copy plasmid for overexpression.</p></td>
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    <td><p id="picTextCenter">Figure 4: Plasmid map of pSBX1K3; low copy plasmid for overexpression.</p></td>
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<h3>References</h3>
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<p> [1] <a name="Ref" href="http://www.addgene.org/vector-database/2657/"> http://www.addgene.org/vector-database/2657/</a> </p>
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Latest revision as of 02:10, 18 October 2014


Plasmids

pETblue

Since a critical part of our project was the overexpression, chosing the correct vector was crucial. Of particular importance are of course the quality of the promotor and the RBS, as well as a His-Tag for purification. All these parts are available in the parts-registry, but even with the convenient cloning of BioBricks, we thought a more conventional approach would suit our needs better. We chose a pET-vector, which we could easily modify by ligating it with annealed oligo nucleotides. The vector pETBlue-1 already came equipped with a T7-promotor and a LacZ site (see figure 1).

Figure 1: Plasmid map of pETBlue-1 [1] . XbaI and XmaI were used to insert the annealed oligo nucleotides to create an easily accessible vector for overexpression.

Figure 2: Product of olio nucleotide annealing for insertion in pETBlue-1.

Using the oligo nucleotides depicted in figure 2, we were able to create a convenient vector for IPTG-inducible overexpression of genes in the RFC25 standard. The aligned oligo nucleotides had sticky ends to ligate with XbaI and EcoRI sites, creating a scar.

The DNA fragment from annealed oligos was to introduce:

  • very strong RBS
  • 6xHis-Tag
  • Thromin-cleavage-site
  • NgoMIV-compatible restriction site for in-frame insertion

The last point proved to be somewhat tricky. Unfortunately, as figure 1 shows, pETBlue-1 is not optimal for RFC25 cloning, since it has both NgoMIV and AgeI restriction sites. We got around this problem by using a third enzyme with the same sticky ends: XmaI. This was to be the up-stream site for insertion. Further modifications of the pET-vector were supposed to include the deletion of all NgoMIV and AgeI sites, as well as the changing of the introduced XmaI site to AgeI, so that the vector could be used with conventional RFC25 enzymes.
The AgeI site was successfully changed using site directed mutagenesis PCR. However, the difficulty we had with expression using the modified pETBlue1.2 led us to abandon this side project.

Heidelberg's pSBX1K3 and pSBX4K5

As part of our collaboration with Team Heidelberg we received two expression vectors they designed, pSBX1K3 and pSBX4K5. We have used pSBX1K3 for test expressions of our constructs.

Figure 3: Plasmid map of pSBX1K3; high copy plasmid for overexpression.

Figure 4: Plasmid map of pSBX1K3; low copy plasmid for overexpression.

References

[1] http://www.addgene.org/vector-database/2657/

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