Difference between revisions of "Part:BBa K3187019"
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<tr>
 
<tr>
 
<td><b>Origin</b></td>
 
<td><b>Origin</b></td>
−
<td>Synthetic</td>
+
<td><i>Synthetic</i></td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
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     <br>LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines)  The molecular weight  
 
     <br>LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines)  The molecular weight  
 
     is 1.478 kDa.
 
     is 1.478 kDa.
 +
</p>
 +
<p>LPETGG was linked to coat protein <a href="https://parts.igem.org/Part:BBa_K3187017"target="_blank">BBa_K3187017</a> that
 +
  build the composite part coat protein with LPETGG tag (CP-LPETGG <a href="https://parts.igem.org/Part:BBa_K3187000"target="_blank">BBa_K3187000</a>)
 +
  and mCherry
 +
  <a href="https://parts.igem.org/Part:BBa_K3187026"target="_blank">BBa_K3187026</a>. Also mCherry with LPETGG tag is a composite part
 +
  <a href="https://parts.igem.org/Part:BBa_K3187009"target="_blank">BBa_K3187009</a>
 +
  The LPETGG tag of coat protein was used to modify the coat protein with GGGG-mCherry and the assembeld Virus-like particles (VLPs).
 +
Furthermore, the tag enables us to control the functionality of Sortase A7M.
 +
This was tested with the coupling of mCherry-LPETGG and GGGG-mCherry <a href="https://parts.igem.org/Part:BBa_K3187008"target="_blank">BBa_K3187008</a>.
 +
</p>
 +
 +
<h3>Results</h3>
 +
<p>The SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry (Fig. 1) shows small red fluorescent bands when the concentration of sortase
 +
  is lower than 1:3. The bands are approximately found by 54 kDa. This suggests that two mCherrys are linked because the molecular
 +
  weight is roughly doubled compared to the weight of one mCherry (27 kDa). Want to know more about the sortase ratio?
 +
  Please look at our <a href="http://2019.igem.org/Team:TU_Darmstadt/Project/Sortase"target="_blank">wiki</a>.
 +
 +
  <div style="text-align: center;">
 +
      <a href="https://2019.igem.org/wiki/images/6/67/T--TU_Darmstadt--SDS_buff_fluor_Srt5_7_1%3B3_1%3B10_.png"target="_blank">
 +
    <img class="img-fluid center" src="https://2019.igem.org/wiki/images/6/67/T--TU_Darmstadt--SDS_buff_fluor_Srt5_7_1%3B3_1%3B10_.png" style="max-width:60%" />
 +
    </a>
 +
   
 +
    <div class="caption">
 +
      <p>
 +
      <b>Figure 1:</b>
 +
            SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry with Sortase A7M.
 +
        </p>
 +
    </div>
 +
    </div>
 +
</p>
 +
<p>Coat proteins with LPETGG assemble to Virus-like particles (VLPs). Images were made with the transmission electron microscopy (TEM)
 +
  and the diameter of VLPs was measured with dynamic light scattering.
 +
  <div style="text-align: center;"> 
 +
      <a href="https://static.igem.org/mediawiki/parts/b/bc/T--TU_Darmstadt--TEM_CP_ohne_SP.jpeg"target="_blank"> 
 +
  <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%" />
 +
    </a>
 +
      <div class="caption">
 +
        <p>
 +
        <b>Figure 3:</b>
 +
              Assembly of only coat proteins with LPETGG. 
 +
          </p>
 +
      </div>
 +
</div>
 +
<p>The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering
 +
    (DLS) analysis.
 +
    <div style="text-align: center;"> 
 +
            <a href="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png"target="_blank">
 +
    <img class="img-fluid center" src="https://2019.igem.org/wiki/images/6/68/T--TU_DARMSTADT--DLS_ohne_Mod.png" style="max-width:60%" />
 +
        </a>
 +
        <div class="caption">
 +
          <p>
 +
          <b>Figure 5:</b>
 +
              Diagram of DLS measurment of VLPs
 +
            </p>
 +
        </div>
 +
    </div>
 +
 +
        As shown in the diagram, VLPs which only consist of  coat proteins with LPETGG tag and VLPs made out of coat proteins
 +
        without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). Since, there is no difference between VLPs with and without LPETGG tag,
 +
        the tag does not disturb the functionality of coat proteins.
 +
        </p>
 
</p>
 
</p>
 
<h2>References</h2>
 
<h2>References</h2>

Revision as of 14:37, 18 October 2019


LPETGG Tag for Sortase-mediated Ligation

Profile

Name LPETGG
Base pairs 18
Molecular weight 1.478 kDa
Origin Synthetic
Part Basic part
Properties Recognition sequence for sortase A

Usage and biology

Generally, the amino acid sequence LPXTG (X can be any amino acid) is a recognition sequence for the Sortase A. It is found in S. aureus and is important for anchoring exoproteins in the peptidoglycan layer. Exoproteins are pathogenicity. LPXTG is recognized by sortase A a transpeptidase which cleaves between the threonine and the glycine. The threonine forms an amid bond with the pentaglycine sequence of the peptidoglycan layer. As a result, a covalently bond is produced. It has been proven that the sequence LPETG is well recognized by sortase A [1] . LPXTG is an easy opportunity to modify proteins, for example with peptides or other proteins that contains a polyG tag. We used a tag with four glycines BBa_K3187018. Thus VLPs are easily modified since they are made of proteins.
LPETGG is also possible recognized by sortase A. We used this sequence, since it was used in the publication, we based our project on. [2]
LPETGG consists of six amino acids (lysine, proline, glutamine acid, threonine and two glycines) The molecular weight is 1.478 kDa.

LPETGG was linked to coat protein BBa_K3187017 that build the composite part coat protein with LPETGG tag (CP-LPETGG BBa_K3187000) and mCherry BBa_K3187026. Also mCherry with LPETGG tag is a composite part BBa_K3187009 The LPETGG tag of coat protein was used to modify the coat protein with GGGG-mCherry and the assembeld Virus-like particles (VLPs). Furthermore, the tag enables us to control the functionality of Sortase A7M. This was tested with the coupling of mCherry-LPETGG and GGGG-mCherry BBa_K3187008.

Results

The SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry (Fig. 1) shows small red fluorescent bands when the concentration of sortase is lower than 1:3. The bands are approximately found by 54 kDa. This suggests that two mCherrys are linked because the molecular weight is roughly doubled compared to the weight of one mCherry (27 kDa). Want to know more about the sortase ratio? Please look at our wiki.

Figure 1: SDS-PAGE of the coupling of mCherry-LPETGG and GGGG-mCherry with Sortase A7M.

Coat proteins with LPETGG assemble to Virus-like particles (VLPs). Images were made with the transmission electron microscopy (TEM) and the diameter of VLPs was measured with dynamic light scattering.

Figure 3: Assembly of only coat proteins with LPETGG.

The diameter of VLPs consisting of different protein combinations was measured with dynamic light scattering (DLS) analysis.

Figure 5: Diagram of DLS measurment of VLPs

As shown in the diagram, VLPs which only consist of coat proteins with LPETGG tag and VLPs made out of coat proteins without a tag are smaller than P22-VLPs containing CP and SP (Fig. 5). Since, there is no difference between VLPs with and without LPETGG tag, the tag does not disturb the functionality of coat proteins.

References

  1. ↑ Silvie Hansenová Maňásková , Kamran Nazmi, Alex van Belkum, Floris J. Bikker, Willem J. B. van Wamel, Enno C. I. Veerman, Synthetic LPETG-Containing Peptide Incorporation in the Staphylococcus aureus Cell-Wall in a Sortase A- and Growth Phase-Dependent Manner, plos one, 19.02.2014 [1]
  2. ↑ Dustin Patterson,*,†Benjamin Schwarz,‡John Avera,‡Brian Western,†Matthew Hicks,†Paul Krugler,†Matthew Terra, †Masaki Uchida,‡Kimberly McCoy,‡and Trevor Douglas*,Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22Virus-like Particle, Bioconjugate Chemistry, 2017, 28, 2114−2124 [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]