Difference between revisions of "Part:BBa K2668020"
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<h2>Introduction</h2>
 
<h2>Introduction</h2>
 
<p>Sirius (BBa_K2668020) is a fusion protein between CBM3a and mRFP1. By fusing CBM3a to a fluorescent moiety, this part allowed us to investigate the binding capability of CBM3a platform to cellulose. </p>
 
<p>Sirius (BBa_K2668020) is a fusion protein between CBM3a and mRFP1. By fusing CBM3a to a fluorescent moiety, this part allowed us to investigate the binding capability of CBM3a platform to cellulose. </p>
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<h2>Construction</h2>
 
<h2>Construction</h2>
 
<p>The Sirius part (BBa_K2668020) correspond to a CBM3a and mRFP1 sequences fused by an endogenous C terminal linker. 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.</p>
 
<p>The Sirius part (BBa_K2668020) correspond to a CBM3a and mRFP1 sequences fused by an endogenous C terminal linker. 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.</p>
  
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                    <b>Figure 1: </b> <b>Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map. </b> 
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                    CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder. 
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<h2>Characterisation</h2>
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<h3>1. Production of Sirius</h3>
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<p>The part BBa_K2668020 was cloned into the pET28 expression vector using In-Fusion Cloning Kit. The resulting construct was transformed into E. coli strain BL21 and expression of the recombinant protein was induced using IPTG.  The His-tagged protein was then purified on IMAC resin charged with cobalt. Results are shown on figure 2. A large amount of protein at the expected size for Sirius (52 kDa, lane CFE) was found predominant in elution samples (E1/40 and E1/100). The degree of purity of full length Sirius was about 72%. In addition to the full length protein, several extra bands that likely correspond to proteolysis products were observed.</p>
 
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                <a href="/File:T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" class="image">
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                    <img alt="" src="/wiki/images/8/80/T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" width="360" height="248" class="thumbimage" /></a> 
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                    <b>Figure 2:</b> <b>SDS-PAGE analysis of Sirius purification fractions </b> 
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                    CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder. 
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<h3>Validation of Sirius</h3>
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<p>Once produced in E. coli, fixation of Sirius to cellulose was tested using pull down assays. 70 µM of Sirius protein or mRFP1 (without CBM3a) or buffer were incubated with cellulose (Avicell).  After several washes with resuspension buffer, cellulose pellets were recovered and the associated fluorescence was measured. Results are shown on figure 3. Only the cellulose pellet incubated with Sirius protein displays a high level of fluorescence. Control experiments showed that only background levels of fluorescence are retained in the cellulose pellet incubated with mRFP1 alone (no CBM3a). These results clearly show that CBM3a of Sirius interacts with cellulose, and thus mediates fixation of the mRFP1 protein to cellulose. </p>
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             <a href="/File:T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" class="image">
 
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                 <b>Figure 2:</b> <b>SDS-PAGE analysis of Sirius purification fractions </b>
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                 <b>Figure 3: </b> <b>Fluorescence retained in the cellulose pellet after pull down (triplicate test) </b>   
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                CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder.  
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<h2>Characterisation</h2>
 
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<h3>1. Production of Sirius</h3>
 
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<p>The part BBa_K2668020 was cloned into the pET28 expression vector using In-Fusion Cloning Kit. The resulting construct was transformed into E. coli strain BL21 and expression of the recombinant protein was induced using IPTG.  The His-tagged protein was then purified on IMAC resin charged with cobalt. Results are shown on figure 2. A large amount of protein at the expected size for Sirius (52 kDa, lane CFE) was found predominant in elution samples (E1/40 and E1/100). The degree of purity of full length Sirius was about 72%. In addition to the full length protein, several extra bands that likely correspond to proteolysis products were observed.</p>
 
  
  

Revision as of 20:12, 9 October 2018


Sirius: CBM3a - mRFP1 fusion

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1272
    Illegal AgeI site found at 1384
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

Sirius (BBa_K2668020) is a fusion protein between CBM3a and mRFP1. By fusing CBM3a to a fluorescent moiety, this part allowed us to investigate the binding capability of CBM3a platform to cellulose.

Construction

The Sirius part (BBa_K2668020) correspond to a CBM3a and mRFP1 sequences fused by an endogenous C terminal linker. 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.

Figure 1: Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map. CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder.

Characterisation

1. Production of Sirius

The part BBa_K2668020 was cloned into the pET28 expression vector using In-Fusion Cloning Kit. The resulting construct was transformed into E. coli strain BL21 and expression of the recombinant protein was induced using IPTG. The His-tagged protein was then purified on IMAC resin charged with cobalt. Results are shown on figure 2. A large amount of protein at the expected size for Sirius (52 kDa, lane CFE) was found predominant in elution samples (E1/40 and E1/100). The degree of purity of full length Sirius was about 72%. In addition to the full length protein, several extra bands that likely correspond to proteolysis products were observed.

Figure 2: SDS-PAGE analysis of Sirius purification fractions CFE: cell free extract, FT: flow through, W: washes, E1/40: elution with 40mM imidazole, E1/100: elution with 100 mM imidazole, E2/100: elution with 100 mM imidazole, E1/300: elution with 300 mM imidazole, MW: molecular weight ladder.

Validation of Sirius

Once produced in E. coli, fixation of Sirius to cellulose was tested using pull down assays. 70 µM of Sirius protein or mRFP1 (without CBM3a) or buffer were incubated with cellulose (Avicell). After several washes with resuspension buffer, cellulose pellets were recovered and the associated fluorescence was measured. Results are shown on figure 3. Only the cellulose pellet incubated with Sirius protein displays a high level of fluorescence. Control experiments showed that only background levels of fluorescence are retained in the cellulose pellet incubated with mRFP1 alone (no CBM3a). These results clearly show that CBM3a of Sirius interacts with cellulose, and thus mediates fixation of the mRFP1 protein to cellulose.

Figure 3: Fluorescence retained in the cellulose pellet after pull down (triplicate test)