Difference between revisions of "Part:BBa K4390063"

Revision as of 16:48, 13 October 2022

CBD-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

The CBD is the non-catalytic domain of cellulase that recognises the beta-1,4-glycosidic linkage of cellulose and cellulose-derived polymers (e.g. carboxymethylcellulose). In the design, we used CenA for our CBD, the cellulose-binding domain of the cellulase endoglucanase A from Cellulomonas fimi (Din, N. et al., 1994). We attached the CBD to the N-terminus of the sfGFP via a glycine-serine linker.

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.

This part encodes sfGFP fused to the cellulose-binding domain (CBD) from the cellulase beta-1,4-endoglucanase of Cellulomonas fimi. The green-fluorescent recombinant protein was used as an indicator to show qualitatively and quantitatively whether the CBD has affinity towards our carboxymethylcellulose-based hydrogels. 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 CBD indeed binds to the carboxymethylcellulose-based hydrogels 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).

From the SDS-PAGE, we were able to tell that the proteins were successfully expressed in BL21(DE3) cells.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 11
Illegal NheI site found at 34
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 450

Reference

Din, N., Forsythe, I., Burtnick, L., Gilkes, N., Miller, R., Warren, R. and Kilburn, D. (1994). The cellulose-binding domain of endoglucanase A (CenA) from Cellulomonas fimi: evidence for the involvement of tryptophan residues in binding. Molecular Microbiology, 11(4), pp.747-755.

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 The CBD is the non-catalytic domain of cellulase that recognises the beta-1,4-glycosidic linkage of cellulose and cellulose-derived polymers (e.g. carboxymethylcellulose). In the design, we used CenA for our CBD, the cellulose-binding domain of the cellulase endoglucanase A from Cellulomonas fimi (Din, N. et al., 1994). We attached the CBD to the N-terminus of the sfGFP via a glycine-serine linker.
-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.
+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.
 This part encodes sfGFP fused to the cellulose-binding domain (CBD) from the cellulase beta-1,4-endoglucanase of Cellulomonas fimi. The green-fluorescent recombinant protein was used as an indicator to show qualitatively and quantitatively whether the CBD has affinity towards our carboxymethylcellulose-based hydrogels. 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 CBD indeed binds to the carboxymethylcellulose-based hydrogels and thus, immobilises the sfGFP fused to it.