Difference between revisions of "Part:BBa K3187008"
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                 <h3>Profile</h3>
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                 <h1>Profile</h1>
 
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                 <h3> Usage and Biology</h3>
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                 <h1> Usage and Biology</h1>
 
                 <p>mCherry <a href="https://parts.igem.org/Part:BBa_K3187026" target="_blank">(BBa_K3187026)</a> is a red
 
                 <p>mCherry <a href="https://parts.igem.org/Part:BBa_K3187026" target="_blank">(BBa_K3187026)</a> is a red
 
                     fluorescent
 
                     fluorescent
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                <h1>Results</h1>
  
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                 <h3> Methods</h3>
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                 <h2>FRET-based assay to analyze Sortase ligation efficiency</h2>
  
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                <h4>Purification</h4>
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  <h2>Comparison of MGGGG-mCherry and GGGG-mCherry (<a href="https://parts.igem.org/Part:BBa_K2868010"
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                <p>The GGGG-mCherry was heterologously expressed in <i>E. coli</i> BL21 and purified with
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                                        target="_blank">BBa_K2868010</a></h2>
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                    <a href="#" target="_blank">GE Healthcare ÄKTA Pure machine</a>
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                    which is a machine for FPLC. The used affinity tag was Strep-tagII.
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                </p>
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                <h4>SDS-Page and Western blot</h4>
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                <p>To verify that the CP-LPETGG was produced, a SDS-Page <a href="#" target="_blank">SDS-Page</a>
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                    followed by a
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                    <a href="#" target="_blank">Western blot</a> was performed.
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                <h3>Results</h3>
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                <h4>Cloning and Expression</h4>
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                <p>The successful cloning was proven with sanger sequencing and production with a Western blot.
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                    <div style="text-align: center;">
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                        <img class="img-fluid center"
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                            src="https://2019.igem.org/wiki/images/4/49/T--TU_Darmstadt--western_blot.png"
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                            style="max-width:60%"/>
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                        <div class="caption">
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                <p>
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                    <b>Figure 1:</b>
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                    Western blot of all produced and purified proteins.
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                </p>
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            </div>
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        <p>Fig. 1 shows that the band of the GGGG-mCherry is approximatley found by the 28.5 kDa band. Consequently, the
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            successful protein production
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            was proven.CP-LPETGG is detected with Strep-Tactin-HRP.</p>
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Revision as of 12:25, 21 October 2019


GGGG-Tag for Sortase-mediated Ligation x mCherry Fluorescence Protein

Profile

Name GGGG-mCherry
Base pairs 1028
Molecular weight 28.5 kDa
Origin synthetic, derived from Discosoma sp.
Parts mCherry, GGGG-sequence, T7 Promoter, lac Operator, GASPAG Linker, 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 (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 pET24 vector, containing the sequence of mCherry, a GGGG-sequence, 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 T7 Terminator (BBa_K3187032), 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]

Results

FRET-based assay to analyze Sortase ligation efficiency

Comparison of MGGGG-mCherry and GGGG-mCherry (BBa_K2868010

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]
Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 854
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 854
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 915
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 854
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 854
  • 1000
    COMPATIBLE WITH RFC[1000]