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, an Amnis ImageStream ISX and TEM with Immunogold labelling and protein expression was determined via SDS-PAGE and Western Blot.
Figure 1 These data from a flow cytometer show the fluorescence profile for wild-type BL21(DE3). The wild type demonstrates no significant fluorescence.
Figure 2 These data from a flow cytometer 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 number 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. The result also suggests that sfGFP is able to move through the pore formed during pilus biosynthesis.
Another way of testing the sfGFP parts was immunogold labelling. This sequence specific method would ideally tell us whether the cell was exporting our modified pili, or just the native pili. It was carried out in the BL21(DE3) strain.
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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