Difference between revisions of "Part:BBa K2668020"
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<partinfo>BBa_K2668020 short</partinfo>
 
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<partinfo>BBa_K2668020 parameters</partinfo>
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<h2>Introduction</h2>
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<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>
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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>
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<figure class="figure"  style="text-align:center;">
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<img style="width : 70%; heigth = auto;" src="" alt="An awesome Agarose gel">
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<figcaption class="figure-caption"><strong>Figure 1:</strong> Figure 1: Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map. </figcaption>
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</figure>
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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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This part results of the fusion of mRFP1 to CBM3a. The aim of this construct is to prove the binding of CBM3a to cellulose and to quantify it by fluorescence. Furthermore, the mRFP1 is visible to the naked eye and can therefore be used as a visual proof of concept
 
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2668020 parameters</partinfo>
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Revision as of 19:35, 9 October 2018


Sirius: CBM3a - mRFP1 fusion

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 class="figure" style="text-align:center;"> <img style="width : 70%; heigth = auto;" src="" alt="An awesome Agarose gel"> <figcaption class="figure-caption">Figure 1: Figure 1: Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map. </figcaption> </figure>

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.


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]