Difference between revisions of "Part:BBa K3187017"
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     <div class="col mx-2">
 
     <div class="col mx-2">
 
      
 
      
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    <h2>coat protein </h2>
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        <h2>Profile</h2>
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    <table style=“width:80%“>
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        <table style=“width:80%“>
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    <tr>
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        <tr>
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    <td>name</td>
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        <td><b>Name</b></td>
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    <td>coat protein </td>
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        <td>Coat protein </td>
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    </tr>
+
        </tr>
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    <tr>
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        <tr>
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    <td>base pairs</td>
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        <td><b>Base pairs</b></td>
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    <td>1293</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>molecular weight</td>
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        <td><b>Molecular weigth</b></td>
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    <td>46.9 kDa</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>origin</td>
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        <td><b>Origin</b></td>
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    <td>bacteriophage P22</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>parts</td>
+
        <td><b>Parts</b></td>
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    <td>coat protein</td>
+
        <td>Basic part</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>
+
        <td><b>Properties</b></td>
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    <td>Assembly with scaffold proteins to VLPs </td>
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        <td>Assembly with scaffold proteins to VLPs </td>
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    </tr>
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        </tr>
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    </table>
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        </table>
 
     <h3> Usage and Biology</h3>
 
     <h3> Usage and Biology</h3>
 
         <p> Coat Protein is an umbrella term for many different proteins, which simplify the transfer  
 
         <p> Coat Protein is an umbrella term for many different proteins, which simplify the transfer  
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                       We focused on the viral and bacteriophagic coat proteins, which are parts of their respective organisms’  capsid.  
 
                       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
 
                       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
+
                       transfer the genetic information into the infected cell.  
 +
                      <sup id="cite_ref-2" class="reference">
 +
                        <a href="#cite_note-1">[2] </a>
 +
                      </sup>
 +
                      Because of the high variety of coat proteins, we are focussing
 
                         on one specific coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">(BBa_K3187017)</a>,  
 
                         on one specific coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">(BBa_K3187017)</a>,  
 
                         which is naturally found in the bacteriophage P22.
 
                         which is naturally found in the bacteriophage P22.
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−
          </p>
+
        </p>
 +
    <h3>Methods</h3>
 +
    <p>The basic part coat protein is  produced and purified as the composite part <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">(BBa_K3187000)</a> (CP-LPETGG)
 +
      and <a href="https://parts.igem.org/Part:BBa_K3187001"target="_blank">(BBa_K3187001)</a> (CP).  For gene expression and
 +
      protein purification as CP-LPETGG the coding sequence contains a coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">(BBa_K3187017)</a>
 +
      a LPETGG <a href="https://parts.igem.org/Part:BBa_K3187019"target="_blank">(BBa_K3187019)</a>,
 +
      a <a href="https://parts.igem.org/Part:BBa_K3187029"target="_blank">T7 promoter, <i>lac</i>-operator and RBS (BBa_K3187029)</a>,
 +
      a Short Linker 5AA <a href="https://parts.igem.org/Part:BBa_K3187030"target="_blank">(BBa_K3187030)</a>
 +
      T7 terminator <a href="https://parts.igem.org/Part:BBa_K3187032"target="_blank">(BBa_K3187032)</a>, and
 +
      Strep-tagII <a href="https://parts.igem.org/Part:BBa_K3187025"target="_blank">(BBa_K3187025)</a>.
 +
      The composite part coat protein without LPETGG <a href="https://parts.igem.org/Part:BBa_K3187001"target="_blank">(BBa_K3187001)</a>
 +
      contains a <a href="https://parts.igem.org/Part:BBa_K3187029"target="_blank">T7 promoter, <i>lac</i>-operator and RBS (BBa_K3187029)</a>,
 +
      two terminators (T7Te terminator and rrnb T1 terminator <a href="https://parts.igem.org/Part:BBa_K3187036"target="_blank">(BBa_K3187036)</a>),
 +
      a Short Linker 5AA <a href="https://parts.igem.org/Part:BBa_K3187030"target="_blank">(BBa_K3187030)</a>
 +
      and Strep-tagII<a href="https://parts.igem.org/Part:BBa_K3187025"target="_blank">(BBa_K3187025)</a>. The production is performed in the <i>E. coli</i>  strain BL21 (DE3)
 +
      and it is purified with <a href="#"target="_blank">GE Healthcare ÄTKA Pure machine</a>which is a machine for FPLC. To verify the successful production, 
 +
      a Western blot is carried out.
 +
    </p>
 
   <h3> Results</h3>
 
   <h3> Results</h3>
 
       <p> The basic part coat portein was expressed, produced and purified as the composite part  
 
       <p> The basic part coat portein was expressed, produced and purified as the composite part  
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         which  
 
         which  
 
         is a machine for FPLC.
 
         is a machine for FPLC.
−
         To verify the right production, a <a herf="#"target="_blank">Western blot</a>is made.
+
         To verify the right production, a <a herf="#"target="_blank">Western blot</a> is made.
 
         <div style="text-align: center;">  
 
         <div style="text-align: center;">  
 
         <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%" />
 
         <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>
 
         </div>
 
         </div>
 
         </div>
−
         <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
+
         <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 production
 
           was proven. CP-LPETGG and CP are detected with Strep-Tactin-HRP.
 
           was proven. CP-LPETGG and CP are detected with Strep-Tactin-HRP.
 
         </p>  
 
         </p>  
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                 <a rel="nofollow" class="external autonumber" href="#">[1] </a>
 
                 <a rel="nofollow" class="external autonumber" href="#">[1] </a>
 
                 </span>
 
                 </span>
 +
              </li>
 +
              <li id="cite_note-2">
 +
                <span class="mw-cite-backlink">
 +
                          <a href="#cite_ref-2">↑</a>
 +
                </span>
 +
                      <span class="reference-text">
 +
                          Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines:
 +
                          Genetics, Structure, and Mechanism, 2005, 80- 88
 +
                          <a rel="nofollow" class="external autonumber"
 +
                              href="https://link.springer.com/chapter/10.1007/0-387-28521-0_5">[2] </a>
 +
                      </span>
 
               </li>
 
               </li>
 
             </ol>
 
             </ol>

Revision as of 05:38, 16 October 2019


P22 Bacteriophage Coat Protein

Profile

Name Coat protein
Base pairs 1293
Molecular weigth 46.9 kDa
Origin Bacteriophage P22
Parts Basic part
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. [2] Because of the high variety of coat proteins, we are focussing on one specific coat protein (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), they assemble to a VLP and form the basis for our modular platform.

Methods

The basic part coat protein is produced and purified as the composite part (BBa_K3187000) (CP-LPETGG) and (BBa_K3187001) (CP). For gene expression and protein purification as CP-LPETGG the coding sequence contains a coat protein (BBa_K3187017) a LPETGG (BBa_K3187019), a T7 promoter, lac-operator and RBS (BBa_K3187029), a Short Linker 5AA (BBa_K3187030) T7 terminator (BBa_K3187032), and Strep-tagII (BBa_K3187025). The composite part coat protein without LPETGG (BBa_K3187001) contains a T7 promoter, lac-operator and RBS (BBa_K3187029), two terminators (T7Te terminator and rrnb T1 terminator (BBa_K3187036)), a Short Linker 5AA (BBa_K3187030) and Strep-tagII(BBa_K3187025). The production is performed in the E. coli strain BL21 (DE3) and it is purified with GE Healthcare ÄTKA Pure machinewhich is a machine for FPLC. To verify the successful production, a Western blot is carried out.

Results

The basic part coat portein was expressed, produced and purified as the composite part BBa_K3187000 (coat protein with LPETGG) BBa_K3187001 (coat protein). The production is performed in E. coliBL21 and it is purified with GE Healthcare ÄKTA Pure machine which is a machine for FPLC. To verify the right production, a Western blot is 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 production 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]
  2. ↑ Sherwood Casjens and Peter Weigele, DNA Packaging by Bacteriophage P22, Viral Genome Packaging Machines: Genetics, Structure, and Mechanism, 2005, 80- 88 [2]
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]