Results

Chassis determination

As we wanted to express modified pili in E. coli we ideally needed a chassis that did not natively produce pili. The genome of E. coli MG1655 is annotated as containing the fim operon (Accession NC_000913) and was therefore proposed to act as a positive control in all experiments. For modified pili production two strains were initially proposed: E. coli Top10 (Invitrogen) and E. coli DH5α (NEB). Top10 is a derivative of DH10B, the genome of which (Accession CP000948) is lacking an annotated fim operon whereas there is no published genome for DH5α. Therefore it was important to verify whether these two strains were lacking the pili producing fim operon.

Using a genomic extraction and PCR we attempted to isolate and amplify three known genes of the fim operon in MG1655 and DH5α. We ran the products of these two preparations down an agarose gel. The results of the electrophoresis procedure showed the same bands in the MG1655 as in the DH5α, representative of three genes in the operon. This demonstrates DH5α's possession of the operon and therefore the unsuitability of DH5α as the chassis for our plasmid inserts.

The other strain identified in the literature as potentially lacking the fim operon was Top10. A similar procedure was used as above, involving a genomic extraction and an enzymatic amplification of the genes of interest. Once more, the MG1655 strain was used as a positive control. The PCR products were run down an agarose gel and the results showed that Top10 does not contain the three genes of interest and so does not have the operon or natively produce pili.

Overall, these results confirmed that MG1655 would be ideal as a positive pili producing control and, that Top10 would be a suitable chassis for our modified pili whereas DH5α would not. In addition two further chassis strains were chosen, the E. coli K-12 FimB knockout strain (JW4275-1 from the Keio collection Baba et al) that the Harvard 2015 iGEM team demonstrated did not produce pili and the E. coli K12 FimH knockout strain (JW4283-3 from the Keio collection Baba et al) which is deficient in the gene that we specifically wanted to modify. Finally, E. coli BL21(DE3) was used to allow initial protein expression to be investigated using the T7 promoter.

Modified Pili Expression

Part construction

The modular cloning strategy (described in the materials and methods) was used an attempt to build plasmids containing either: the fim operon under two different promoters; or six modified versions of fimH (and wild type fimH) under for different promoters. The results of our cloning attempts are given in Table 1.

Part testing

FimH_sfGFP expression

The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). This part produces a FimH adhesin protein fused with sfGFP (Pedelaq et al 2005) 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.

Figure 3 Data from the plate reader showing fluorescence intensity for WT-BL21(DE3) and T7_FimH_225_sfGFP, average from n=3 and error bars show standard deviation of the mean.These results show that fluorescence from cultures containing T7_FimH_225_sfGFP is higher than WT-BL21(DE3). This shows that FimH_225_sfGFP is expressed and does give rise to fluorescence. According to standard curves made in the lab on the same equipment and settings, we produced a 130 ng/ml concentration of sfGFP in the sample.

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. Moving on from this result, we were confident that the fusion of metal binding domains to the FimH protein would not negatively affect pilus formation as our chosen domains, and other potentials that may make up a modular toolkit, were smaller than sfGFP .

Figure 5 : Western blot of BL21 WT and BL21 with our T7-FimH_225_GFP construct, 1-5: sfGFP, BL21 WT soluble fraction, BL21 WT pellet, BL21 with T7-FimH_225_GFP solluble fraction, BL21 with T7-FimH_225_GFP pellet

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.

We were unable to detect fluorescence in cells that had the FimH fusion protein under control of the rhamnose inducible promoter despite experimenting with the induction protocol over a range of growth temperatures and rhamnose concentrations . Given the relatively low proportion of cells in the overall culture of the T7_FimH_225_sfGFP that showed significant fluorescence, it is perhaps unsurprising that a weaker and less well characterised promoter(than T7) should not give strong results. Our consequent decision was to attempt to test the rhamnose inducible promoter by other means. We used a fusion of 6xhistine tag and FimH as an alternative reporter mechanism with the aim of establishing whether modified FimH constructs could be expressed with P_Rha.

FimH_1_His expression

We therefore looked to express FimH with a HisTag at the first amino acid position under control of P_Rha.

Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein without characterising the resultant part. This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. The coding sequence is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the fim operon As well as being utilised as a metal binding protein, this part also acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of E. coli strains (BL21(DE3), Top10, ΔFimB, and ΔFimH), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be probed by the use of an SDS-PAGE and a Western blot.

The results of an SDS-PAGE and subsequent Western blot provided bands at the corresponding molecular weights for the FimH_1His protein which also had binding affinities for an anti-His antibody. The result of a band that corresponds to a molecular weight marginally lighter than the entire intact FimH_1His evidentiated expression of the protein, and suggested that the signal peptide had been cleaved upon the proteins delivery to the cell surface membrane. This makes a case for successful export of the protein and therefore successful pilus biogenesis.

Fim Operon expression

Applied Design

Hydrocyclone

Metal Binding Reactor

Biosecurity