Difference between revisions of "Part:BBa K2668070"
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
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<p>The part BBa_K2668070 was used to validate the Orthos part (i.e. fusion protein between CBM3a and a monomeric streptavidin head). </p>
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<p>The part BBa_K2668070was used to validate the streptavidin head of the Cerberus platform (</html><partinfo>BBa_K2668010</partinfo><html>). </p>
 
<h2>Construction</h2>
 
<h2>Construction</h2>
 
<p>The part BBa_K2668070 correspond to genetic sequence encoding for mTag Blue Fluorescent Protein fused with an AviTag. IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by infusion into the pSB1C3 plasmid and then transformed into E. coli Dh5-alpha strain. Figure 1 shows the restriction map of the resulting clones. We expected two bands at 700bp. All the clones show the expected restriction profiles. The clones 2 was deposited in the registry of Standard Biological Parts.</p>
 
<p>The part BBa_K2668070 correspond to genetic sequence encoding for mTag Blue Fluorescent Protein fused with an AviTag. IDT performed the DNA synthesis and delivered the part as gBlock.  The construct was cloned by infusion into the pSB1C3 plasmid and then transformed into E. coli Dh5-alpha strain. Figure 1 shows the restriction map of the resulting clones. We expected two bands at 700bp. All the clones show the expected restriction profiles. The clones 2 was deposited in the registry of Standard Biological Parts.</p>
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         </div>
 
         </div>
 
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     </div>
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<h2>Characterisation</h2>
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<h2>Characterization</h2>
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<p>The part BBa_K2668070 was used to assess the ability of Orthos to functionalize cellulose with a compound biotinylated in vivo. Indeed, proteins containing the AviTag small peptide can be efficiently biotinylated invivo by BirA. The part BBa_K2668070 was cloned into the pETDuet-1 expression vector previously cloned with BirA  and transformed  into E. coli strain BL21. Biotin was added in the culture medium (0.1 mM final concentration) during IPTG induction, in order to produce biotinylated BFP. Next, 3.2 µM of Orthos were incubated with 32 µM of in vivo biotinylated BFP. For the control experiment the same quantity of BFP was incubated without Orthos. This samples were finally incubated with cellulose. After several washes with resuspension buffer, fluorescence was measured in the cellulose pellets. As shown in Figure 2, fluorescence is two time higher in the sample containing Orthos than in the sample than in the control sample.</p>
+
<p>The part BBa_K2668070 was used to assess the ability of a chimeric fusion between the cellulose binding domain CBM3a and a monomeric streptavidin head mSA2 (see part </html><partinfo>BBa_K2668010</partinfo><html>), a construction we nicknamed Orthos, to functionalize cellulose with a compound biotinylated <em>in vivo</em>. Indeed, proteins containing the AviTag small peptide can be efficiently biotinylated invivo by BirA. BirA is the <em>E. coli</em> biotin ligase that has high affinity toward Avi Tag, a short peptidic sequence. The part BBa_K2668070 was cloned into the pETDuet-1 expression vector previously cloned with BirA  and transformed  into <em>E. coli</em> strain BL21. Biotin was added in the culture medium (0.1 mM final concentration) during IPTG induction, in order to produce biotinylated BFP. Next, 3.2 µM of Orthos were incubated with 32 µM of in vivo biotinylated BFP. For the control experiment the same quantity of BFP was incubated without Orthos. This samples were finally incubated with cellulose. After several washes with resuspension buffer, fluorescence was measured in the cellulose pellets. As shown in Figure 2, fluorescence is two time higher in the sample containing Orthos than in the sample than in the control sample.</p>
 
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<p>These results demonstrate that Orthos protein is able to bind to a compound biotinylated in vivo. In addition, they show that Orthos binds to cellulose and thus provide a proof of principle that Orthos design allows functionalization of cellulose with a fluorophore through its streptavidin linker.</p>
+
<p>These results demonstrate that the mTagBFP blue fluorescent protein was well-tagged with biotine which validate the part. In addition, they show that Orthos binds to cellulose and thus provide a proof of principle that Orthos design allows functionalization of cellulose with a fluorophore through its streptavidin linker.</p>
  
 
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Revision as of 19:45, 10 October 2018


mTagBFP AviTag 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]


Introduction

The part BBa_K2668070was used to validate the streptavidin head of the Cerberus platform (BBa_K2668010).

Construction

The part BBa_K2668070 correspond to genetic sequence encoding for mTag Blue Fluorescent Protein fused with an AviTag. IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by infusion into the pSB1C3 plasmid and then transformed into E. coli Dh5-alpha strain. Figure 1 shows the restriction map of the resulting clones. We expected two bands at 700bp. All the clones show the expected restriction profiles. The clones 2 was deposited in the registry of Standard Biological Parts.

Figure 1: Analyses of pSB1C3_ mTagBFP and AviTag length and restriction map The plasmids of 5 obtained clones were analysed to check their length. XbaI/NcoI digested plasmids are electrophoresed through a 1% agarose gel. Lane 1 & 2 and 4 to 6 are the digested plasmids (clones 1 to 5) resulting from DNA extraction of the 5 clones. Lane 3 is the Smart DNA ladder (Eurogentec), the 0.6kb, 1.5 kb and 2.5kb DNA fragments are annotated.

Characterization

The part BBa_K2668070 was used to assess the ability of a chimeric fusion between the cellulose binding domain CBM3a and a monomeric streptavidin head mSA2 (see part BBa_K2668010), a construction we nicknamed Orthos, to functionalize cellulose with a compound biotinylated in vivo. Indeed, proteins containing the AviTag small peptide can be efficiently biotinylated invivo by BirA. BirA is the E. coli biotin ligase that has high affinity toward Avi Tag, a short peptidic sequence. The part BBa_K2668070 was cloned into the pETDuet-1 expression vector previously cloned with BirA and transformed into E. coli strain BL21. Biotin was added in the culture medium (0.1 mM final concentration) during IPTG induction, in order to produce biotinylated BFP. Next, 3.2 µM of Orthos were incubated with 32 µM of in vivo biotinylated BFP. For the control experiment the same quantity of BFP was incubated without Orthos. This samples were finally incubated with cellulose. After several washes with resuspension buffer, fluorescence was measured in the cellulose pellets. As shown in Figure 2, fluorescence is two time higher in the sample containing Orthos than in the sample than in the control sample.

Figure 2: Fluorescence remaining on cellulose fraction after several washes Ctrl BFP: BFP witout Orthos; Orthos BFP: BPP + Orthos. (*Mann Whitney test p-value 0.01)

These results demonstrate that the mTagBFP blue fluorescent protein was well-tagged with biotine which validate the part. In addition, they show that Orthos binds to cellulose and thus provide a proof of principle that Orthos design allows functionalization of cellulose with a fluorophore through its streptavidin linker.