Difference between revisions of "Part:BBa K2324013"

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pRha_FimH_1His

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). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein. 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. Harvard iGEM 2105 introduced a SpyTag at position 1 https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850004 but were unable to provide characterisation of this part. Therefore we decided to change the SpyTag for a 6x-Histidine tag to allow for easy characterisation of expression via SDS-PAGE and Western Blot. 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 remaining coding sequences from the fim operon. </br> 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.

After 24 h growth, cultures containing the P_Rha_FimH1His and wild-type strains were harvested. Cells were disrupted using BugBuster protein extraction reagent (Merck) and samples of the soluble and insoluble fractions were prepared for SDS-PAGE. Western Blots were probed with an anti-6xHis primary antibody raised in Mouse and anti-Mouse alkaline phosphatase-conjugated secondary antibody and visualised with SigmaFast BCIP/NBT.

Results

Figure 1 Image of SDS-PAGE gel stained with Coomassie Blue. The wells of the polyacrylamide gel contained the following, 1: marker, 2: ΔFimH with FimH_1His insoluble fraction, 3: ΔFimB with FimH_1His insoluble fraction, 4: Top10 with FimH_1His insoluble fraction, 5: ΔFimH with FimH_1His soluble fraction, 6: ΔFimH with FimH_1His soluble fraction, 7: Top10 with FimH with FimH_1His soluble fraction, 8: BL21(DE3) with FimH_1His soluble fraction, 9: ΔFimB with FimH_1His soluble fraction and 10: marker.

Figure 2 Image of SDS-PAGE gel stained with Coomassie Blue. The wells is this gel are contained the following: 0: marker, 1: WT-BL21(DE3) insoluble fraction , 2: WT-Top10 insoluble fraction, 3: WT-ΔFimB insoluble fraction, 4: WT-ΔFimH insoluble fraction, 5: WT-BL21(DE3) soluble fraction, 6: WT-Top10 soluble fraction, 7: WT-ΔFimB soluble fraction, 8: WT-ΔFimH soluble fraction and 9: BL21(DE3) with FimH_1His insoluble fraction.

Figure 3 The columns of the Western blot correspond to the following samples: 0: marker 1: ΔFimB with FimH_1His insoluble fraction, 2: Top10 with FimH_1His insoluble fraction, 3: BL21(DE3) with FimH_1His insoluble fraction, 4: BL21(DE3) with FimH_1His insoluble fraction, 5: BL21(DE3) with FimH_1His soluble fraction, 6: ΔFimH with FimH_1His soluble fraction, 7: ΔFimB with FimH_1His soluble fraction, 8: Top10 with FimH with FimH_1His soluble fraction and 9: marker.

The images of the SDS-PAGE gels demonstrate that the cells were successfully disrupted as protein bands were seen in both soluble and insoluble fractions. However, due to the fact that the samples were crude protein extracts, the gel images do not definitively show whether FimH-1His is expressed. The Western Blot image shows bands at the corresponding molecular weights for the FimH_1His protein with (32 kDa) and without (30 kDa) the signal peptide. In the ΔFimH culture, a band is seen in the soluble fraction corresponding to the molecular weight of the un-cleaved signal peptide-FimH_1His protein. This indicates that the protein is expressed but remains in the cytoplasm of the cell. In the BL21(DE3), Top10 and ΔFimB cultures, bands are seen in the insoluble fraction corresponding to the molecular weight of the cleaved FimH_1His protein. The lack of signal peptide demonstrates that the protein has been exported from the cytoplasm and the fact that the bands only appear in the insoluble fraction suggests that FimH-1His is somehow associated with the cell membrane. It is unclear as to why export should not occur in the ΔFimH strain.

Conclusion

The results from the Western-Blot have demonstrated successful expression of FimH_1His from the rhamnose inducible promoter in all four E. coli strains. The results also suggest in three out of the four strains that the protein is exported from the cell. In order to determine if FimH_1His can associate with the rest of the fim operon proteins further work is required either in BL21(DE3) (a pili producing strain) or strains which co-harbour our P_Ara-fim operon or P_J23100-fim operon https://parts.igem.org/Part:BBa_K2324012.

References

Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655. Pallesen, L., Poulsen, L. K., Christiansen, G., and Klemm, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848. Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88. Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.

Sequence and Features