Difference between revisions of "Part:BBa K2668020"
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<partinfo>BBa_K2668020 short</partinfo>
 
<partinfo>BBa_K2668020 short</partinfo>
 
<partinfo>BBa_K2668020 parameters</partinfo>
 
<partinfo>BBa_K2668020 parameters</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2668020 SequenceAndFeatures</partinfo>
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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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<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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<img style="width : 70%; heigth = auto;" src="https://static.igem.org/mediawiki/parts/8/80/T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" alt="An awesome Agarose gel"/><br/><strong>Figure 1:</strong> Figure 1: Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map.
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            <a href="/File:T--INSA-UPS_France--CqsA.png" class="image">
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                <img alt="" src="/wiki/images/thumb/c/cb/T--INSA-UPS_France--CqsA.png/800px-T--INSA-UPS_France--CqsA.png" width="800" height="auto" class="thumbimage" />
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            <b>Figure 2:</b> <b>SDS-PAGE analysis of Sirius purification fractions </b>
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<h2>Characterisation</h2>
 
<h2>Characterisation</h2>
 
<h3>1. Production of Sirius</h3>
 
<h3>1. Production of Sirius</h3>
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Revision as of 19:52, 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.


</p> 

           <a href="/File:T--INSA-UPS_France--CqsA.png" class="image">
               <img alt="" src="/wiki/images/thumb/c/cb/T--INSA-UPS_France--CqsA.png/800px-T--INSA-UPS_France--CqsA.png" width="800" height="auto" class="thumbimage"  />
           </a>  

           Figure 2: SDS-PAGE analysis of Sirius purification fractions

Characterisation

1. Production of Sirius

<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.