Difference between revisions of "Part:BBa K3187017"
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<h2>LPETGG</h2>
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<div class="container">
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<table style=“width:80%“>
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  <div class="row">
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<tr>
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    <div class="col mx-2">
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<td>name</td>
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<td>LPETGG</td>
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    <h2>coat protein </h2>
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</tr>
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    <table style=“width:80%“>
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<tr>
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    <tr>
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<td>basepairs</td>
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    <td>name</td>
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<td>18</td>
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    <td>coat protein </td>
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</tr>
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    </tr>
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<tr>
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    <tr>
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<td>molecular weight</td>
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    <td>base pairs</td>
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<td>1478 Da</td>
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    <td>1293</td>
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</tr>
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    </tr>
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<tr>
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    <tr>
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<td>origin</td>
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    <td>molecular weight</td>
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<td><i>synthetic</i></td>
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    <td>46.9 kDa</td>
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</tr>
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    </tr>
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<tr>
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    <tr>
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<td>parts</td>
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    <td>origin</td>
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<td>basic part</td>
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    <td>bacteriophage P22</td>
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</tr>
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    </tr>
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<tr>
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    <tr>
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<td>properties</td>
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    <td>parts</td>
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<td>recognition sequence for sortase A </td>
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    <td>coat protein</td>
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</tr>
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    </tr>
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</table>
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    <tr>
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    <td>properties</td>
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    <td>Assembly with scaffold proteins to VLPs </td>
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    </tr>
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    </table>
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    <h3> Usage and Biology</h3>
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        <p> Coat Protein is an umbrella term for many different proteins, which simplify the transfer
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          of molecules between different compartments that are surrounded by a membrane.
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                <sup id="cite_ref-1" class="reference">
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                        <a href="#cite_note-1">[1] </a>
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                      </sup>
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                      We focused on the viral and bacteriophagic coat proteins, which are parts of their respective organisms’  capsid.
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                      The genetic information (DNA or RNA) is wrapped and protected by the capsid. During cell infection, the phage or virus
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                      transfer the genetic information into the infected cell. Because of the high variety of coat proteins, we are focussing
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                        on one specific coat protein (BBa_K3187017)  <a herf="https://parts.igem.org/Part:BBa_K3187017"target="_blank">BBa_K3187017</a>,
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                        which is naturally found in the bacteriophage P22.
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                      This coat protein (CP) consists of 431 amino acids and its molecular weight is 46.9 kDa.  Because of its significance as
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                      a part of the
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                      capsid, it represents one main part of our Virus-like particle. Together with the scaffold protein
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                      (BBa_K3187021)  <a herf="https://parts.igem.org/Part:BBa_K3187021"target="_blank">BBa_K3187021</a>,
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                      they assemble to a VLP and form the basis for our modular platform.
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          </p>
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  <h3> Results</h3>
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      <p> The basic part coat portein was expressed, produced and purified as the composite part BBa_K3187000 (coat protein with LPETGG)
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          <a herf="https://parts.igem.org/Part:BBa_K3187000"target="_blank">BBa_K3187000</a> and BBa_K3187001 (coat protein)
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          <a herf="https://parts.igem.org/Part:BBa_K3187001"target="_blank">BBa_K3187001</a>.
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        The production is performed in <i>E. coli</i>BL21 and it is purified with GE Healthcare ÄKTA Pure machine
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        which
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        is a machine for FPLC<a herf="#"target="_blank">GE Healthcare ÄKTA Pure machine LINK which is a machine for FPLC</a>.
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        To verify the right production, a Western blot <a herf="#"target="_blank">Western blot</a>is made.
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        <div style="text-align: center;">
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        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/9/9c/T--TU_Darmstadt--Western_blot_CP-LPETGG_CP.jpeg" style="max-width:60%" />
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        <div class="caption">
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          <p>
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          <b>Figure 1:</b>
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                Western blot of all produced and purified proteins.
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            </p>
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        </div>
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        </div>
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        <p>Fig. 1 shows that the band of the CP-LPETGG is approximately found by the 49 kDa band and the band of CP by 46.9 kDa. Consequently, the successful cloning and expression
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          was proven. CP-LPETGG and CP are detected with Strep-Tactin-HRP.
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        </p>
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      </p>  
  
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<h4>Usage and biology</h4>
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<p>Generally, the amino acid sequence LPXTG, where X can be every amino acid, is a recognition sequence. It is found in
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         <h2>References</h2>
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    <i>S. aureus</i> and it is important for anchoring exoproteins in the peptidoglycan layer. Exoproteins are responsible for
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            <ol class="references">
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    pathogenicity of the organism. LPXTG is recognized by sortase A, a transpeptidase which cleaves between the
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              <li id="cite_note-1">
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    threonine (T) and the glycine (G). The threonine forms an amide bond with the pentaglycine sequence of the peptidoglycan
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                <span class="mw-cite-backlink">
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    layer. So, a covalent bond between protein and the outside of the peptidoglycan layer is produced. It has been shown that
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                  <a href="#cite_ref-1">↑</a>
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    the sequence LPETG is well recognized by sortase A
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                </span>
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    <sup id="cite_ref-1" class="reference">
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                <span class="reference-text">
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            <a href="#cite_note-1">[1] </a>
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                  Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport,  
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          </sup>.
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                  NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414
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    LPXTG is an easy opportunity to modify proteins, for example with peptides or other proteins that contains a polyG tag.
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                <a rel="nofollow" class="external autonumber" href="#">[1] </a>
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    We used a tag with four glycines <a href="https://parts.igem.org/Part:BBa_K3187018"target="_blank">BBa_K3187018</a>.
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                </span>
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    As a result, VLPs are easily modified too, because VLPs are made of proteins.
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              </li>
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    <br>LPETGG can also be employed as a recognition sequence. The sequence shown below was derived from our main literature source
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             </ol>
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    <sup id="cite_ref-2" class="reference">
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    </div>
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         <a href="#cite_note-2">[2] </a>
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  </div>
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    </sup>
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</div>
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    on which our project is based on. <br>LPETGG consists of 18 DNA base pairs and translates to a six amino acid sequence
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    containing lysine, proline, glutamine acid, threonine and two glycines. The molecular weight of the peptide is 1478 Da.
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</p>
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<h2>References</h2>
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<ol class="references">
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  <li id="cite_note-1">
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    <span class="mw-cite-backlink">
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      <a href="#cite_ref-1">↑</a>
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    </span>
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    <span class="reference-text">
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            Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman,  
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            Synthetic LPETG-Containing Peptide Incorporation in the <i>Staphylococcus aureus</i> Cell-Wall in a Sortase A- and Growth
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            Phase-Dependent Manner, plos one, 19.02.2014
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    <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260 ">[1] </a>
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    </span>
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  </li>
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+
−
    <li id="cite_note-2">
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          <span class="mw-cite-backlink">
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−
             <a href="#cite_ref-2">↑</a>
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          </span>
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          <span class="reference-text">
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−
                Dustin Patterson,*,†Benjamin Schwarz,‡John Avera,‡Brian Western,†Matthew Hicks,†Paul Krugler,†Matthew Terra,
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                †Masaki Uchida,‡Kimberly McCoy,‡and Trevor Douglas*,Sortase-Mediated Ligation as a Modular Approach for the
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                Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22Virus-like Particle, Bioconjugate
+
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                Chemistry, 2017, 28, 2114−2124
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          <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1371/journal.pone.0089260 ">[2] </a>
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          </span>
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        </li>
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Revision as of 07:41, 15 October 2019


P22 Bacteriophage Coat Protein

coat protein

name coat protein
base pairs 1293
molecular weight 46.9 kDa
origin bacteriophage P22
parts coat protein
properties Assembly with scaffold proteins to VLPs

Usage and Biology

Coat Protein is an umbrella term for many different proteins, which simplify the transfer of molecules between different compartments that are surrounded by a membrane. [1] We focused on the viral and bacteriophagic coat proteins, which are parts of their respective organisms’ capsid. The genetic information (DNA or RNA) is wrapped and protected by the capsid. During cell infection, the phage or virus transfer the genetic information into the infected cell. Because of the high variety of coat proteins, we are focussing on one specific coat protein (BBa_K3187017) BBa_K3187017, which is naturally found in the bacteriophage P22. This coat protein (CP) consists of 431 amino acids and its molecular weight is 46.9 kDa. Because of its significance as a part of the capsid, it represents one main part of our Virus-like particle. Together with the scaffold protein (BBa_K3187021) BBa_K3187021, they assemble to a VLP and form the basis for our modular platform.

Results

The basic part coat portein was expressed, produced and purified as the composite part BBa_K3187000 (coat protein with LPETGG) BBa_K3187000 and BBa_K3187001 (coat protein) BBa_K3187001. The production is performed in E. coliBL21 and it is purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLCGE Healthcare ÄKTA Pure machine LINK which is a machine for FPLC. To verify the right production, a Western blot Western blotis made.

Figure 1: Western blot of all produced and purified proteins.

Fig. 1 shows that the band of the CP-LPETGG is approximately found by the 49 kDa band and the band of CP by 46.9 kDa. Consequently, the successful cloning and expression was proven. CP-LPETGG and CP are detected with Strep-Tactin-HRP.

References

  1. ↑ Juan S. Bonifacino, Jennifer Lippincott-Schwartz, Coat proteins: shaping membranetransport, NATURE REVIEWS MOLECULAR CELLBIOLOGY, May 2013, 4, 409-414 [1]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]