Difference between revisions of "Part:BBa K3187004"

 
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__NOTOC__
 
<partinfo>BBa_K3187004 short</partinfo>
 
<partinfo>BBa_K3187004 short</partinfo>
 +
<html>
 +
 +
                <h3>Profile</h3>
 +
                <table style=“width:80%“>
 +
                <tr>
 +
                <td><b>Name</b></td>
 +
                <td>TEV site-polyG-scaffold protein</td>
 +
                </tr>
 +
 +
                <tr>
 +
                <td><b>Base pairs</b></td>
 +
                <td> 1028</td>
 +
                </tr>
 +
 +
                <tr>
 +
                <td><b>Molecular weight</b></td>
 +
                <td>27.8 kDa</td>
 +
                </tr>
 +
 +
                <tr>
 +
                <td><b>Origin</b></td>
 +
                <td> Tabacco Etch Virus (TEV); <i>Aequorea victoria</i></td>
 +
                </tr>
 +
 +
                <tr>
 +
                <td><b>Parts</b></td>
 +
                <td>T7-Promoter, lac-operator, RBS (g10 leader sequence), TEV protease recognition sequence, polyG, sfGFP, Strep-tag II, T7 terminator</td>
 +
                </tr>
 +
                <tr>
 +
                <td><b>Properties</b></td>
 +
                <td> After cleavage by the TEV protease, the polyG tag can be used to fuse sfGFP to the Sortase A recognition sequence(LPTEGG) </td>
 +
                </tr>
 +
                </table>
 +
                <h3> Usage and Biology</h3>
 +
            <p>
 +
 +
 +
The TEV protease is cleaving a protein after a specific sequence between Glutamine and Serine or Glycine
 +
 +
<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> .
 +
 +
We are using this to create a free N-terminal polyG sequence in front of sfGFP so we can use it as substrate in a Sortase A mediated reaction
 +
 +
<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>
 +
 +
<sup id="cite_ref-5" class="reference">
 +
                            <a href="#cite_note-5">[5]
 +
                            </a>
 +
                    </sup> .
 +
<br>
 +
 +
sfGFP is a variant of the fluorescence protein GFP that was originally isolated from the jellyfish <i>Aequorea victoria</i>. It has a short maturing time of 13.6&nbsp;min, has an extinction maximum at 485&nbsp;nm and an emission maximum at 510&nbsp;nm.
 +
 +
<sup id="cite_ref-6" class="reference">
 +
                            <a href="#cite_note-6">[6]
 +
                            </a>
 +
                    </sup>
 +
 +
<sup id="cite_ref-7" class="reference">
 +
                            <a href="#cite_note-7">[7]
 +
                            </a>
 +
                    </sup> 
 +
<br>
 +
At the end of the sfGFP a strep tag was added to enable easy protein purification.
 +
<br>
 +
The part contains a T7 promoter so it can be transcribed by T7 polymerase, and a lac operator so protein expression can be induced by IPTG.
 +
</p>
 +
 +
 +
 +
 +
 +
               
 +
             
 +
                </p>
 +
               
 +
                <h3> Methods</h3>
 +
                <h4>Cloning</h4>
 +
                <p>The fusion protein was cloned into the pACYC2 backbone with <a href="#"target="_blank">Gibson Assembly</a> . To verify the cloning,
 +
                    the sequence was controlled by sanger sequencing by Microsynth Seqlab.
 +
                </p>
 +
                <h4>Purification</h4>
 +
                <p>The protein was heterologously expressed in <i>E.&nbsp;coli</i> BL21 and purified with
 +
                    <a href="#"target="_blank">GE Healthcare ÄKTA FPLC</a>. The used affinity tag was Strep-tag II.
 +
                </p>
 +
                <h4>SDS-Page and Western blot</h4>
 +
                <p>To verify that the CP-LPETGG was produced, a <a href="#"target="_blank">SDS-Page</a> followed by a
 +
                    <a href="#"target="_blank">Western blot</a> was performed.
 +
                </p>
 +
               
 +
 +
                </p>
 +
                <h4>Assembly</h4>
 +
                <p> The assembly is tested <i>in vivo</i> and <i>in vitro</i>. The assembled VLPs are collected with
 +
                    <a href="#"target="_blank">ultracentrifugation</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>Cloning and Expression</h4>
 +
                <p>The successful cloning was proven with sanger sequencing and production with a Western blot.
 +
                        <div style="text-align: center;"> 
 +
                        <img class="img-fluid center" src="https://2019.igem.org/wiki/images/1/1a/T--TU_Darmstadt--westernplot_sp.jpeg" style="max-width:60%" />
 +
                        <div class="caption">
 +
                          <p>
 +
                          <b>Figure 1:</b>
 +
                                Western blot of all produced and purified proteins.
 +
                            </p>
 +
                        </div>
 +
                        </div>
 +
                <p>Fig. 1 shows that SP has a molecular weight of approximatley 45&nbsp;kDa. This is about the expected size of 46.1&nbsp;kDa. Two additional bands in this lane can be observed. One at est. 25 kDa and one between 25 and 37 kDa. The lower band may be sfGFP and upper band scaffold protein. We came to this conclusion by comparing the lane of Sp-sfGFP with lanes of only SP and with sfGFP with TEV cleavage site. Those two bands are probably produced by the denaturing of the SP-sfGFP fusion protein. During denaturation for SDS-PAGE sample preparation, the fusion protein can break in two parts, sometimes it breaks in front and sometimes after the Strep-tag. This is indicated by the fact that both bands are stained by an anti strep-tag western blot.
 +
The band of Strep-tag and SP can be observed at a size of est. 30 kDa. This is larger than the expected, theoretical size of SP at about 18 kDa. Because the plasmid used for expression was verified by sequencing before, and the fusion protein has the right size when it is not broken from sample preparation, we suspect that the protein is the right one and it just behaves unexpected in this SDS PAGE.
 +
 +
                  </p>       
 +
 +
                                         
 +
                     
 +
                </p>
 +
               
 +
               
 +
                </p>
 +
 +
                <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
 +
                        treatment, 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> The image shows assembled VLPs. The green flourescence of the VLP pellet indictes that we succesfully loaded our VLPs with sfGFP by using our sfGFP-SP fusion protein.
 +
 +
 +
            </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-23>↑</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">[3] </a>
 +
                            </span>
 +
                        </li>
 +
                    </ol>
 +
 +
</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 15:45, 16 October 2019


TEV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X Superfolder Green Fluorescence Protein

Profile

Name TEV site-polyG-scaffold protein
Base pairs 1028
Molecular weight 27.8 kDa
Origin Tabacco Etch Virus (TEV); Aequorea victoria
Parts T7-Promoter, lac-operator, RBS (g10 leader sequence), TEV protease recognition sequence, polyG, sfGFP, Strep-tag II, T7 terminator
Properties After cleavage by the TEV protease, the polyG tag can be used to fuse sfGFP to the Sortase A recognition sequence(LPTEGG)

Usage and Biology

The TEV protease is cleaving a protein after a specific sequence between Glutamine and Serine or Glycine [1] [2] . We are using this to create a free N-terminal polyG sequence in front of sfGFP so we can use it as substrate in a Sortase A mediated reaction [3] [4] [5] .
sfGFP is a variant of the fluorescence protein GFP that was originally isolated from the jellyfish Aequorea victoria. It has a short maturing time of 13.6 min, has an extinction maximum at 485 nm and an emission maximum at 510 nm. [6] [7]
At the end of the sfGFP a strep tag was added to enable easy protein purification.
The part contains a T7 promoter so it can be transcribed by T7 polymerase, and a lac operator so protein expression can be induced by IPTG.

Methods

Cloning

The fusion protein was cloned into the pACYC2 backbone with Gibson Assembly . To verify the cloning, the sequence was controlled by sanger sequencing by Microsynth Seqlab.

Purification

The protein was heterologously expressed in E. coli BL21 and purified with GE Healthcare ÄKTA FPLC. The used affinity tag was Strep-tag II.

SDS-Page and Western blot

To verify that the CP-LPETGG was produced, a SDS-Page followed by a Western blot was performed.

Assembly

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

Results

Cloning and Expression

The successful cloning was proven with sanger sequencing and production with a Western blot.

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

Fig. 1 shows that SP has a molecular weight of approximatley 45 kDa. This is about the expected size of 46.1 kDa. Two additional bands in this lane can be observed. One at est. 25 kDa and one between 25 and 37 kDa. The lower band may be sfGFP and upper band scaffold protein. We came to this conclusion by comparing the lane of Sp-sfGFP with lanes of only SP and with sfGFP with TEV cleavage site. Those two bands are probably produced by the denaturing of the SP-sfGFP fusion protein. During denaturation for SDS-PAGE sample preparation, the fusion protein can break in two parts, sometimes it breaks in front and sometimes after the Strep-tag. This is indicated by the fact that both bands are stained by an anti strep-tag western blot. The band of Strep-tag and SP can be observed at a size of est. 30 kDa. This is larger than the expected, theoretical size of SP at about 18 kDa. Because the plasmid used for expression was verified by sequencing before, and the fusion protein has the right size when it is not broken from sample preparation, we suspect that the protein is the right one and it just behaves unexpected in this SDS PAGE.

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 treatment, 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 image shows assembled VLPs. The green flourescence of the VLP pellet indictes that we succesfully loaded our VLPs with sfGFP by using our sfGFP-SP fusion protein.

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. 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 [3]


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 840
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 138