Part:BBa_K4390027
SB7-tagged sfGFP
This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.
Usage and Biology
SB7 is a 7-amino-acid silica-binding motif from the spore coat protein CotB1 of Bacillus cereus. According to studies, its three arginine residues are important for binding to silica surfaces. We tagged this sequence to the N-terminus of sfGFP. Therefore, when the lysate from a cell culture expressing SB7-tagged sfGFP is mixed with silica beads followed by washing off the supernatant, we would be left with green-coloured silica beads which indicates that sfGFP is bound to them. This is one of the three types of silica-binding tags we explored for immobilising proteins to silica beads.
The green fluorescent protein (GFP) is a protein that can produce bright green colour. Due to these properties, it has always been used as reporter of protein expression. Protein fused with sfGFP will turn the colony green if expressed, which can be used to identify the ability of certain genes to be expressed in other organism like E. coli. Superfolder GFP (sfGFP) is a more robustly folded version of GFP, which is derived from GFP that often misfolds when expressed as fusions with other proteins. Superfolder GPF shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics.
The green-fluorescent recombinant protein was used as an indicator to show qualitatively and quantitatively whether the SB7 peptide has affinity towards silica beads. This was assayed by comparing the green fluorescence intensity between the initial lysate and the supernatant after addition of the silica beads. There should be a significant reduction in fluorescence intensity to prove that the SB7 peptide indeed binds to silica beads and thus, immobilises the sfGFP fused to it.
Characterization
After protein expression in the BL21(DE3) cell cultures, the cultures were lysed by sonication, and the lysates were run on an SDS-PAGE gel to confirm the presence of our CBD fused proteins (Figure 1). This SDS-PAGE step also serves as a solubility test to confirm that all of our desired proteins are in the soluble fraction and can be properly incorporated into protein immobilisation methods.
Figure 2. SDS-PAGE gel of the lysates containing our expressed constructs. Lane 0 represents the negative control, which is the BL21(DE3) strain containing only the pJUMP29 LacZ acceptor plasmid without any insert. The red lines indicate the bands representing our constructs. The ladder we used was Prestained Protein Marker, Broad Range (7-175 kDa) (NEB #P7708S).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 11
Illegal NheI site found at 34
Illegal NotI site found at 73 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 111
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