Difference between revisions of "Part:BBa K2324011"

Revision as of 18:26, 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 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 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.

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_225sfGFP; and Lane 5 Insoluble fraction of T7_FimH_225sfGFP.

These results show that FimH-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-225sfGFP attached to pili.

TEM with Immunogold labelling results

The Western-Blot indicated that FimH-225sfGFP 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 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 5 The gold particles appear to align with the pili on the cell surface.

Sequence and Features