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            <b>Figure 2:</b> <b>SDS-PAGE analysis of Sirius purification fractions </b>  
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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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Revision as of 20:04, 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.


           <a href="/File:T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" class="image">
               <img alt="" src="/wiki/images/8/80/T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" width="360" height="248" class="thumbimage" /></a>  

                   <a href="/File:T--Toulouse-INSA-UPS--Registry--Youn--SiriusPurif.png" class="internal" title="Enlarge"></a>

               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.


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.