Difference between revisions of "Part:BBa K3187008"
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− | __NOTOC__ | + | __NOTOC__ |
− | <partinfo> | + | <partinfo>BBa_K3187000 short</partinfo> |
− | |||
− | < | + | <html> |
− | === | + | <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> | ||
− | |||
− | |||
− | |||
+ | <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> | + | <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.
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1491
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]