Team:Freiburg/parts/improvement

From 2013.igem.org

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<p> We decided to improve the BioBrick BBa_K128001 coding for the HA-tag – a tag which is commonly used to detect proteins. As it is cloned to all of our designed devices, we were in need of a well-functioning and easy-clonable HA-part. </p>
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<p> We decided to improve the BioBrick BBa_K128001 coding for the HA-tag – this tag facilitates detection, purification and isolation of recombinant proteins. As it is cloned to all of our designed devices, we were in need of a well-functioning and easy-clonable HA-part. </p>
<p> The BBa_K128001 contains the RFC23 pre- and suffix (silver standard) which can be used for the construction of in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also suitable for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix. </p>
<p> The BBa_K128001 contains the RFC23 pre- and suffix (silver standard) which can be used for the construction of in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also suitable for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix. </p>
<p> That’s why we designed a new version of the HA-tag BBa_K128001 with an identical sequence of amino acids but with an RFC25 pre- and suffix. This has following advantages:
<p> That’s why we designed a new version of the HA-tag BBa_K128001 with an identical sequence of amino acids but with an RFC25 pre- and suffix. This has following advantages:
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<p> As tags are often fused at the end (C- or N-terminal) of proteins, this HA-tag facilitates cloning.  </p>
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<p>To maintain the function of the proteins in most cases a N- or C-terminal fusion of the HA-tag has to be performed. With our optimized Biobrick it is possible to create such fusion proteins.  </p>
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<td> <img class="gelpic" src=" https://static.igem.org/mediawiki/parts/d/d7/HA_neu_2_Freigem_2013.png"width="100%"> </td>
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<td <b> Fig.1: BBa_K1150016 with RFC25 prefix and suffix. </b> </td>
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<td> <b > Figure 1: BBa_K1150016 with RFC25 prefix and suffix. </b> </td>
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<p> Before bringing this tag into the iGEM-standard we first tested its functionality via Western blot detection, see Fig.2. As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was possible, see Fig.3. </p>
 
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<p> Before bringing this tag into the iGEM-standard we first tested its functionality via Western blot detection <span id="refer"> <a href="#Fig2"> (Fig.2)</a></span>. As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was possible <span id="refer"> <a href="#Fig3"> (Fig.3) </a></span>. </p>
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<td <b> Fig.2: HA-tag fused to CRY2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-TOPO plasmid.</b></td>
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<td> <b> Figure 2: Western Blot analysis of HA-Cry2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-Topo plasmid via anti-HA antibody.</b></td>
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<p> <td <b> Fig.3: HA-tag fused to different dcas9-fusion proteins encoded on the RFC25 pSB1C3 backbone.</b></td></p>
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<p> <td> <b > Figure 3: Western Blot analysis of different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone via anti-HA antibody and anti-&#946; Actin as loading control.</b><br>
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HEK-293T cells were transfected with standardized dCas9-effector fusion constructs and lysed 42h later. </td></p>
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Latest revision as of 01:53, 5 October 2013


Improvement of a Registry part

We decided to improve the BioBrick BBa_K128001 coding for the HA-tag – this tag facilitates detection, purification and isolation of recombinant proteins. As it is cloned to all of our designed devices, we were in need of a well-functioning and easy-clonable HA-part.

The BBa_K128001 contains the RFC23 pre- and suffix (silver standard) which can be used for the construction of in-frame-fusion parts. In contrast to the RFC25 biobrick (which is also suitable for functional part fusions) there is no start codon and stop codon automatically included in the prefix and suffix.

That’s why we designed a new version of the HA-tag BBa_K128001 with an identical sequence of amino acids but with an RFC25 pre- and suffix. This has following advantages:

  • Cloning the HA-tag BBa_K1150016 N-terminal to other fusion parts leads to an automatic transcriptional start due to the introduced ATG in the RFC25 prefix.
  • Cloning the HA-tag BBa_K1150016 C-terminal to other fusion parts leads to an automatic transcriptional stop due to the introduced stop codon in the RFC25 suffix.

To maintain the function of the proteins in most cases a N- or C-terminal fusion of the HA-tag has to be performed. With our optimized Biobrick it is possible to create such fusion proteins.

Figure 1: BBa_K1150016 with RFC25 prefix and suffix.

Before bringing this tag into the iGEM-standard we first tested its functionality via Western blot detection (Fig.2). As it worked fine in mammalian cells, we used this sequence for our standardized constructs. Testing of our standardized devices including the BBa_K1150016 worked well and also western blot detection was possible (Fig.3) .

Figure 2: Western Blot analysis of HA-Cry2-C-RAF-CIBN-protein encoded on the pEF6/V5-His-Topo plasmid via anti-HA antibody.

Figure 3: Western Blot analysis of different dCas9-fusion proteins encoded on the RFC25 pSB1C3 backbone via anti-HA antibody and anti-β Actin as loading control.
HEK-293T cells were transfected with standardized dCas9-effector fusion constructs and lysed 42h later.

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