Difference between revisions of "Part:BBa K3187008"

 
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    <partinfo>BBa_K3187000 short</partinfo>
  
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<!-- Add more about the biology of this part here
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    <html>
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===Usage and Biology===
+
    <div class="container">
 +
        <div class="row">
 +
            <div class="col mx-2">
 +
                <h3>Profile</h3>
 +
                <table style=“width:80%“>
 +
                    <tr>
 +
                        <td>Name</td>
 +
                        <td>mCherry-LPETGG</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Base pairs</td>
 +
                        <td>1028</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Molecular weight</td>
 +
                        <td>28.5 kDa</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Origin</td>
 +
                        <td>synthetic, derived from <i>Discosoma</i>sp.</td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Parts</td>
 +
                        <td>mCherry, T7-promoter, GGGG-sequence, lac-operator, GASPAG, Strep-Tag II, T7-terminator <br>
 +
                        </td>
 +
                    </tr>
 +
                    <tr>
 +
                        <td>Properties</td>
 +
                        <td> Red fluorescent, Ex λ: 587nm, Em λ: 610 nm</td>
 +
                    </tr>
 +
                </table>
 +
                <br>
 +
                <h3> Usage and Biology</h3>
 +
                <p>mCherry <a href="https://parts.igem.org/Part:BBa_K3187026" target="_blank">(BBa_K3187026)</a>is a red
 +
                    fluorescent
 +
                    protein.
 +
                    Which is a synthetic protein derived from <i>Discosoma</i> sp. by
 +
                    directed evolution. The N-terminal GGGG-sequence <a href="https://parts.igem.org/Part:BBa_K3187018"
 +
                                                                        target="_blank">(BBa_K3187018)</a>
 +
                    can be fused to a protein with a C-terminal LPETGG-Sortase A link <a
 +
                            href="https://parts.igem.org/Part:BBa_K3187019">target="_blank">(BBa_K3187019)</a>
 +
                    by Sortase A. We use mCherry as an easily imaged reporter for checking if the coupling worked.<br>
 +
                    The coding sequence was cloned in pDest vector, containing the sequence of mCherry, a GASPAG-Linker
 +
                    <a
 +
                            href="https://parts.igem.org/Part:BBa_K3187038" target="_blank">(BBa_K3187038)</a>, a
 +
                    Strep-Tag II <a href="https://parts.igem.org/Part:BBa_K3187025" target="_blank">(BBa_K3187025)</a>
 +
                    for
 +
                    Purification, a T7 promoter with lac-operator and an RBS <a
 +
                            href="https://parts.igem.org/Part:BBa_K3187029"
 +
                            target="_blank">(BBa_K3187029)</a>, a T7TE terminator <a
 +
                            href="https://parts.igem.org/Part:BBa_K3187032" target="_blank">(BBa_K3187032)</a>, a
 +
                    GGGG-sequence, a Start-Codon <a href="https://parts.igem.org/Part:BBa_J70593" target="_blank">(BBa_J70593)</a>
 +
                    and a Stop-Codon <a href="https://parts.igem.org/Part:BBa_K2868029"
 +
                                        target="_blank">(BBa_K2868029)</a>(). The
 +
                    coding sequence consists of 851 bp which are translated to 260 amino acids.<sup id="cite_ref-1"
 +
                                                                                                    class="reference"><a
 +
                            href="#cite_note-1">[1] </a> </sup> <br>
 +
                </p>
  
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K3187008 SequenceAndFeatures</partinfo>
 
  
 +
                <h3> Methods</h3>
  
−
<!-- Uncomment this to enable Functional Parameter display  
+
                <h4>Purification</h4>
−
===Functional Parameters===
+
                <p>The GGGG-mCherry was heterologously expressed in <i>E. coli</i> BL21 and purified with
−
<partinfo>BBa_K3187008 parameters</partinfo>
+
                    <a href="#" target="_blank">GE Healthcare ÄKTA Pure machine</a>
−
<!-- -->
+
                    which is a machine for FPLC. The used affinity tag was Strep-tagII.
 +
                </p>
 +
                <h4>SDS-Page and Western blot</h4>
 +
                <p>To verify that the CP-LPETGG was produced, a SDS-Page <a href="#" target="_blank">SDS-Page</a>
 +
                    followed by a
 +
                    <a href="#" target="_blank">Western blot</a> was performed.
 +
                </p>
 +
 
 +
 
 +
                <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/4/49/T--TU_Darmstadt--western_blot.png"
 +
                            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 the band of the GGGG-mCherry is approximatley found by the 28.5 kDa band. Consequently, the
 +
            successful protein production
 +
            was proven.CP-LPETGG is detected with Strep-Tactin-HRP.</p>
 +
 
 +
 
 +
        </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">
 +
                Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572
 +
                <a rel="nofollow" class="external autonumber" href="#https://www.nature.com/articles/nbt1037">[1] </a>
 +
            </span>
 +
            </li>
 +
        </ol>
 +
 
 +
    </div>
 +
    </div>
 +
    </div>
 +
    </html>
 +
 
 +
 
 +
    <!-- Add more about the biology of this part here
 +
    ===Usage and Biology===
 +
 
 +
    <!-- -->
 +
    <span class='h3bb'>Sequence and Features</span>
 +
    <partinfo>BBa_K3187000 SequenceAndFeatures</partinfo>
 +
 
 +
 
 +
    <!-- Uncomment this to enable Functional Parameter display
 +
    ===Functional Parameters===
 +
    <partinfo>BBa_K3187000 parameters</partinfo>
 +
    <!-- -->

Revision as of 08:47, 16 October 2019


   P22 Bacteriophage Coat Protein with LPETGG Tag for Sortase-mediated Ligation



   
    

        

            

                
Profile

                

                    

                        Name
                        mCherry-LPETGG
                    

                    

                        Base pairs
                        1028
                    

                    

                        Molecular weight
                        28.5 kDa
                    

                    

                        Origin
                        synthetic, derived from Discosomasp.
                    

                    

                        Parts
                        mCherry, T7-promoter, GGGG-sequence, lac-operator, GASPAG, Strep-Tag II, T7-terminator 

                        		
                    

                    

                        Properties
                         Red fluorescent, Ex λ: 587nm, Em λ: 610 nm
                    

                

                

                
 Usage and Biology

                
mCherry (BBa_K3187026)is a red
                    fluorescent
                    protein.
                    Which is a synthetic protein derived from Discosoma sp. by
                    directed evolution. The N-terminal GGGG-sequence (BBa_K3187018)
                    can be fused to a protein with a C-terminal LPETGG-Sortase A link target="_blank">(BBa_K3187019)
                    by Sortase A. We use mCherry as an easily imaged reporter for checking if the coupling worked.

                    The coding sequence was cloned in pDest vector, containing the sequence of mCherry, a GASPAG-Linker
                    (BBa_K3187038), a
                    Strep-Tag II (BBa_K3187025)
                    for
                    Purification, a T7 promoter with lac-operator and an RBS (BBa_K3187029), a T7TE terminator (BBa_K3187032), a
                    GGGG-sequence, a Start-Codon (BBa_J70593)
                    and a Stop-Codon (BBa_K2868029)(). The
                    coding sequence consists of 851 bp which are translated to 260 amino acids.[1]   

                



                
 Methods


                
Purification

                
The GGGG-mCherry was heterologously expressed in E. coli BL21 and purified with
                    GE Healthcare ÄKTA Pure machine
                    which is a machine for FPLC. The used affinity tag was Strep-tagII.
                

                
SDS-Page and Western blot

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



                
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 the band of the GGGG-mCherry is approximatley found by the 28.5 kDa band. Consequently, the
            successful protein production
            was proven.CP-LPETGG is detected with Strep-Tactin-HRP.



        

        
References

        
    
            
  1. 
         
             ↑
          
                
                Nathan Shaner, Robert Campbell, Paul Steinbach, Ben Giepmans, Amy Palmer and Roger Tsien, Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, Nature Biotechnology, 2004, 22: 1567-1572
                [1] 
            
            
    2. 
        


    

    

    

    



   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 1491
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]