Part:BBa_K2324011
T7_FimH_225sfGFP
This part produces a FimH adhesin protein fused with sfGFP at its 225th amino acid residue, after signal peptide cleavage. Expression is under the control of an IPTG-inducible, T7 promoter (BBa_I712074), with BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a fluorescent FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the fim operon.The T7 promoter should give very strong expression and sfGFP should both give a visual indication of successful expression and folding. As a large protein, sfGFP would push the chaperone-usher pathway to its steric limits.
We have expressed this construct in BL21(DE3). Fluorescence was measured using a plate reader (Tecan) and an Amnis ImageStream ISX. Protein expression was determined via SDS-PAGE and Western Blot and TEM with Immunogold labelling.
Amnis ImageStream TMX results
Figure 1 Data from the Amnis ImageStream TMX show the fluorescence profile for wild-type BL21(DE3). The wild type demonstrates no significant fluorescence.
Figure 2 Data from Amnis ImageStream TMX show the fluorescence profile for BL21(DE3) with T7_FimH_225sfGFP. This construct shows a strong fluorescent signal in the highlighted portion of the cell and it is a significant difference compared to the wild type.
These results show that a proportion of cells in the overall culture produced strong fluorescence. This fluorescence suggests successful folding of the sfGFP which can be taken as evidence by proxy of FimH folding.
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TEM with Immunogold labelling results
To determine whether any FimH-225sfGFP was exported from the cell and forming pili TEM with immunogold labelling was attempted.
Figure 3 When BL21(DE3) were transformed with T7_FimH_225_sfGFP, it appears to specifically bind the gold particles as shown. The images suggest successful expression and export of these modified FimH proteins and specific binding.
Figure 4 The gold particles appear to align with the pili on the cell surface.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 451
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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