Difference between revisions of "Part:BBa K3187021"

 
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<html>
 +
 
 +
                <h3>Profile</h3>
 +
                <table style=“width:80%“>
 +
                <tr>
 +
                <td><b>Name</b></td>
 +
                <td>Scaffold protein </td>
 +
                </tr>
 +
 
 +
                <tr>
 +
                <td><b>Base pairs</b></td>
 +
                <td> 489</td>
 +
                </tr>
 +
 
 +
                <tr>
 +
                <td><b>Molecular weight</b></td>
 +
                <td>18 kDa</td>
 +
                </tr>
 +
 
 +
                <tr>
 +
                <td><b>Origin</b></td>
 +
                <td> <i>Enterobacteria phage P22</i></td>
 +
                </tr>
 +
 
 +
           
 +
                <tr>
 +
                <td><b>Properties</b></td>
 +
                <td> In combination with the coat protein <a href="https://parts.igem.org/Part:BBa_K3187017">(BBa_K3187017)</a> this protein builds the virus capsid of the P22 phage. </td>
 +
                </tr>
 +
                </table>
 +
 
 +
 
 +
 
 +
                <h3> Usage and Biology</h3>
 +
               
 +
                <p> <p>
 +
The P22 scaffold protein (SP) is an important part of the<i> Enterobacteria phage P22</i> capsid.  The virus capsid is assembled with the help of up to 300 copies of the 18 kDa scaffold protein out of approx. 400 copies of the 47kDa coat protein
 +
 
 +
<sup id="cite_ref-1" class="reference">
 +
                            <a href="#cite_note-1">[1]
 +
                            </a>
 +
                    </sup>
 +
 
 +
 
 +
<sup id="cite_ref-2" class="reference">
 +
                            <a href="#cite_note-2">[2]
 +
                            </a>
 +
                    </sup>.
 +
<br>
 +
After the assembly of the virus-capsid the SP is released into the capsid. In case of a functional P22 bacteriophage, this protein is extracted out of the capsid <i>in vivo</i> while the viral DNA is loaded into the capsid
 +
 
 +
 
 +
<sup id="cite_ref-3" class="reference">
 +
                            <a href="#cite_note-3">[3]
 +
                            </a>
 +
                    </sup>
 +
 
 +
 
 +
<sup id="cite_ref-4" class="reference">
 +
                            <a href="#cite_note-4">[4]
 +
                            </a>
 +
                    </sup> . Because the artificial capsid is not filled with DNA the SP remains in the capsid. By fusing the SP with a cargo-protein, one can load the capsid with said cargo
 +
 
 +
 
 +
<sup id="cite_ref-5" class="reference">
 +
                            <a href="#cite_note-5">[5]
 +
                            </a>
 +
                    </sup>
 +
 
 +
. This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly [6].
 +
                </p>
 +
               
 +
 
 +
 
 +
 
 +
                <h3> Methods</h3>
 +
             
 +
                <h4>Assembly</h4>
 +
                <p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with
 +
                    ultracentrifugation  <a href="#"target="_blank">ultracentrifugatione</a> and are visualized with
 +
                    <a href="#"target="_blank">TEM</a>. For more information look at our <a href="#"target="_blank">wiki</a>
 +
             
 +
 
 +
                <h3>Results</h3>
 +
 
 +
               
 +
 
 +
                <h4> Assembly</h4>
 +
                <p> The images of ultracentrifugation displays that monomeric proteins were separated from assembled capsids by
 +
                        ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation
 +
                        reatment, sediment was clearly visible in the centrifuge tube which we suspected to mainly contain VLPs.  
 +
                        Transmission electron microscopy (TEM) was used to image capsids taken from the sediment. For increased
 +
                        contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually
 +
                        intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly,
 +
                        visit our <a href="#"target="_blank">wiki</a>.
 +
                        <div style="text-align: center;"> 
 +
                        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/5/52/T--TU_DARMSTADT--invitro_UZ_TEM.png" style="max-width:60%" />
 +
                          <div class="caption">
 +
                          <p>
 +
                          <b>Figure 2:</b> Ultracentrifugation of assembled VLPs
 +
                               
 +
                            </p>
 +
                          </div>
 +
                        </div>
 +
                </p>
 +
                <p> The images of TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. As a result,  
 +
                    the CPs produced using this part are fully functional . The CPs assemble with
 +
                    scaffold proteins (SPs) and they can be modified on the surface (Fig. 4). Moreover, CPs also assemble without SPs
 +
                    (Fig. 3).
 +
                </p>
 +
                <div style="text-align: center;"> 
 +
 
 +
                    <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg" style="max-width:40%" />
 +
                     
 +
                        <div class="caption">
 +
                          <p>
 +
                          <b>Figure 3:</b>
 +
                                Assembly of only coat proteins with LPETGG. 
 +
                            </p>
 +
                        </div>
 +
                </div>
 +
                    <p>Fig. 3 shows that no scaffold proteins are necessary for assembly.</p>   
 +
                   
 +
                    <div style="text-align: center;"> 
 +
                    <img class="img-fluid center" src="https://static.igem.org/mediawiki/parts/b/b7/T--TU_Darmstadt--TEM_CP_SP_sGFP.jpeg" style="max-width:40%" />
 +
                       
 +
                        <div class="caption">
 +
                          <p>
 +
                          <b>Figure 4:</b>
 +
                                Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.
 +
                            </p>
 +
                        </div>
 +
                    </div>
 +
                   
 +
                    <p>Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and CP-LPETGG can be modified for this process</p>   
 +
 
 +
 
 +
 
 +
                    <h2>References</h2>
 +
 
 +
 
 +
                    <ol class="references">
 +
                        <li id="cite_note-1">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-1">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                            W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387.
 +
                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1016/0022-2836(76)90278-3">[1] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
 
 +
                   
 +
                        <li id="cite_note-2">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-2">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                                W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nat. Struct. Biol. 2003, 10, 131.
 +
                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1038/nsb891">[2] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
                        <li id="cite_note-3">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-3">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                              King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. 
 +
                            <a rel="nofollow" class="external autonumber" href="https://www.nature.com/articles/251112a0">[3] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
 
 +
<li id="cite_note-4">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-4">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                                S. Casjens and R. Hendrix, (1988) "Control mechanisms in dsDNA bacteriophage assembly", in The Bacteriophages, volume 1, ed. R. Calendar, Plenum Press, p. 15-91.
 +
                            <a rel="nofollow" class="external autonumber" href="https://link.springer.com/chapter/10.1007/978-1-4684-5424-6_2">[4] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<li id="cite_note-5">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-5">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                                Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas, Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle
 +
,ACS Chemical Biology 2014 9 (2), 359-365
 +
                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1021/cb4006529">[5] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
 
 +
<li id="cite_note-6">
 +
                            <span class="mw-cite-backlink">
 +
                                <a href="#cite_ref-6">↑</a>
 +
                            </span>
 +
                            <span class="reference-text">
 +
                                P. R. Weigele, L. Sampson, D. Winn‐Stapley, S. R. Casjens, Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains
 +
, J. Mol. Biol. 2005, 348, 831.
 +
                            <a rel="nofollow" class="external autonumber" href="https://doi.org/10.1016/j.jmb.2005.03.004">[6] </a>
 +
                            </span>
 +
                        </li>
 +
 
 +
 
 +
 
 +
                    </ol>
 +
                   
 +
           
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</html>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 12:26, 16 October 2019


P22 Bacteriophage Scaffolding Protein

Profile

Name Scaffold protein
Base pairs 489
Molecular weight 18 kDa
Origin Enterobacteria phage P22
Properties In combination with the coat protein (BBa_K3187017) this protein builds the virus capsid of the P22 phage.

Usage and Biology

The P22 scaffold protein (SP) is an important part of the Enterobacteria phage P22 capsid. The virus capsid is assembled with the help of up to 300 copies of the 18 kDa scaffold protein out of approx. 400 copies of the 47kDa coat protein [1] [2] .
After the assembly of the virus-capsid the SP is released into the capsid. In case of a functional P22 bacteriophage, this protein is extracted out of the capsid in vivo while the viral DNA is loaded into the capsid [3] [4] . Because the artificial capsid is not filled with DNA the SP remains in the capsid. By fusing the SP with a cargo-protein, one can load the capsid with said cargo [5] . This fusion has to occur at the N-Terminus of the SP, because the C-Terminus is important for mechanism of the assembly [6].

Methods

Assembly

The assembly is tested in vivo and in vitro. The assembled VLPs are collected with ultracentrifugation ultracentrifugatione and are visualized with TEM. For more information look at our wiki

Results

Assembly

The images of ultracentrifugation displays that monomeric proteins were separated from assembled capsids by ultracentrifugation at 150.000 x g in a sucrose cushion (35% w/v). After completion of the ultracentrifugation reatment, sediment was clearly visible in the centrifuge tube which we suspected to mainly contain VLPs. Transmission electron microscopy (TEM) was used to image capsids taken from the sediment. For increased contrast, samples were negative-stained with uranyl acetate. We were able to show a high density of visually intact VLPs all over the sample measuring a diameter of 60 nm or less (Fig. 2). For more information about VLP assembly, visit our wiki.

Figure 2: Ultracentrifugation of assembled VLPs

The images of TEM show the assembled VLPs. VLPs only assemble with functional coat proteins. As a result, the CPs produced using this part are fully functional . The CPs assemble with scaffold proteins (SPs) and they can be modified on the surface (Fig. 4). Moreover, CPs also assemble without SPs (Fig. 3).

Figure 3: Assembly of only coat proteins with LPETGG.

Fig. 3 shows that no scaffold proteins are necessary for assembly.

Figure 4: Assembly of modified CP-LPETGG and scaffold proteins. Several CP-LPETGG are linked to sGFP.

Fig. 4 shows that CP-LPETGG and SPs assemble to VLPs and CP-LPETGG can be modified for this process

References

  1. ↑ W. Earnshaw, S. Casjens, S. C. Harrison, Assembly of the head of bacteriophage P22: X-ray diffraction from heads, proheads and related structures J. Mol. Biol. 1976, 104, 387. [1]
  2. ↑ W. Jiang, Z. Li, Z. Zhang, M. L. Baker, P. E. Prevelige, W. Chiu, Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions, Nat. Struct. Biol. 2003, 10, 131. [2]
  3. ↑ King, J., and Casjens, S. (1974). Catalytic head assembling protein in virus morphogenesis. Nature 251:112-119. [3]
  4. ↑ S. Casjens and R. Hendrix, (1988) "Control mechanisms in dsDNA bacteriophage assembly", in The Bacteriophages, volume 1, ed. R. Calendar, Plenum Press, p. 15-91. [4]
  5. ↑ Dustin P. Patterson, Benjamin Schwarz, Ryan S. Waters, Tomas Gedeon, and Trevor Douglas, Encapsulation of an Enzyme Cascade within the Bacteriophage P22 Virus-Like Particle ,ACS Chemical Biology 2014 9 (2), 359-365 [5]
  6. ↑ P. R. Weigele, L. Sampson, D. Winn‐Stapley, S. R. Casjens, Molecular Genetics of Bacteriophage P22 Scaffolding Protein's Functional Domains , J. Mol. Biol. 2005, 348, 831. [6]


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 351
  • 1000
    COMPATIBLE WITH RFC[1000]