Difference between revisions of "Part:BBa K2324011"

Revision as of 18:34, 1 November 2017

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_K1614000), with BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a fluorescent FimH protein that should initiate 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 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_225_sfGFP. 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.

Western Blot results

Figure 3 Image of a Western-Blot probed with Anti-GFP primary antibody raised in Mouse and Anti-mouse alkaline phosphatase conjugated secondary antibody. Lane 1 Pure GFP; Lane 2 Soluble fraction from WT-BL21(DE3); Lane 3 Insoluble fraction from WT-BL21(DE3); Lane 4 Soluble fraction from T7_FimH_225_sfGFP; and Lane 5 Insoluble fraction of T7_FimH_225_sfGFP.

These results show that FimH-225_sfGFP is expressed and is found in the soluble fraction of the cells. Therefore we conclude that the majority of fluorescence observed is in the cytoplasm of the cell and not from FimH-225_sfGFP attached to pili.

TEM with Immunogold labelling results

The Western-Blot indicated that FimH-225_sfGFP was mainly in the cytoplasm but to determine whether any proportion of expressed FimH-225sfGFP was exported from the cell and forming pili, TEM with immunogold labelling was attempted.

Figure 4 Image from TEM with Immunogold labelling. Gold particle labelling is clearly seen in the structures surrounding the cell suggesting that FimH_225_sfGFP, is exported from the cell.

Figure 5 Image from TEM with Immunogold labelling. The gold particles appear to align with the pili on the cell surface suggesting that FimH_225_sfGFP is able to form pili on the cell surface.

These results suggest that FimH-225_sfGFP is expressed is able to form pili. However WT-BL21(DE3) did show some evidence of labelling of structures surrounding the cells (data not shown) therefore these results are inconclusive.

Conclusion

FimH_225_sfGFP has been successfully expressed from the T7 promoter as seen from the plate reader, ImageStream and Western Blots. TEM with Immunogold images suggest that at least some FimH_225_sfGFP is exported from the cell and forms pili but when compared to WT-BL21(DE3) the results are inconclusive and further work is required to properly investigate this.

Sequence and Features