Part:BBa_K2668020
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.
<img style="width : 70%; heigth = auto;" src="" alt="An awesome Agarose gel"/>
Figure 1: Figure 1: Analyses of pSB1C3_ CBM3a and mRFP1 length and restriction map.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1272
Illegal AgeI site found at 1384 - 1000COMPATIBLE WITH RFC[1000]
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