Team:Exeter/Basic Part

Basic Parts

Most of our basic parts are derived from the terminal protein in pili structures FimH. FimH is expressed as a 300 amino acid protein which is then processed by the removal of a signal peptide, Figure 1. The mature form of the protein (279 amino acids) consists of two domains: the mannose binding domain and the pilin domain (Le Tong et al 2010). The literature shows that FimH can be successfully modified by introducing heterologous protein segments at positions 225 and 258 of the mature protein (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. We decided to construct a suite of FimH modified sequences to not only test a variety of metal binding proteins but also determine, via GFP expression, which position 1 (22), 225 or 258 would be best.

Figure 1: This arrow represents the linear amino acid sequence of the protein FimH. As shown, it can be split into its three essential components. The signal peptide guides the protein to the cell surface membrane and is cleaved upon export. The mannose binding domain is the seat of the bacteria's pathogenicity, its ability to bind to glycosylated cell surface proteins in mammals. The pilin binding domain interacts allosterically with the mannose binding domain and other fim proteins during binding and pilus biogenesis.

FimH sfGFP

To easily monitor expression we chose to modify FimH by inserting the super folder green fluorescent protein (sfGFP) (Pedelacq et al 2005). The sfGFP was inserted in three different places in the FimH protein to act as a reporter on the level of expression of the protein and thus giving an indication of its efficiency. The three places of insertion were 22, which is the place where the signal peptide ends (Hanson et al 1988) in the mannose binding domain, the 225 and 258, which are present in the domain that interacts with FimG.

Name	Description	Base Pairs
BBa_K2324002	FimH_1_sfGFP	1617
BBa_K2324001	FimH_225_sfGFP	1617
BBa_K2324003	FimH_258_sfGFP	1617

FimH Fusion Proteins

We initially chose three metal binding proteins in order to bind a variety of metal ions: Mouse Metallothionein (Huang et al 1981) to bind Cd, Cu and Zn; Synechococcus Metallothionein (Blindauer et al 2001) to bind cadmium and zinc; and Synechococcus Plastocyanin (Inoue et al 1999) to bind copper. We also chose to insert a 6x histidine tag to act as both a reporter of expression but also to bind Ni. Unfortunately no construct was successfully built containing plastocyanin.

Name	Description	Base Pairs
BBa_K2324014	FimH_1_His	921
BBa_K2324004	FimH_1_SynMT	1071
BBa_K2324005	FimH_1_MouseMT	1086

Fim Operon

The fim operon consists of six proteins and their native RBS sequences. For synthesis the operon was split into three separate parts. It was produced by a previous iGEM team from Harvard in 2015.

Name	Description	Base Pairs
BBa_K2324016	FimAIC	1985
BBa_K2324017	FimD	2703
BBa_K2324018	FimFG	1056

References

Blindauer, C. A., Harrison, M. D., Parkinson, J. A., Robinson, A. K., Cavet, J. S., Robinson, N. J., and Sadler, P. J. (2001) A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity. Proc. Natl. Acad. Sci. U.S.A. 98, 9593–9598.

Inoue, T., Sugawara, H., Hamanaka, S., Tsukui, H., Suzuki, E., Kohzuma, T., and Kai, Y. (1999) Crystal Structure Determinations of Oxidized and Reduced Plastocyanin from the Cyanobacterium Synechococcus sp. PCC 7942. Biochemistry 38, 6063–6069.

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.

Huang, I-Y., Kimura, M., Hata A., Tsunoo H., Yoshida A. (1981) Complete Amino Acid Sequence of Mouse Liver Metallothionein-II. Biochemistry 89, 1839-1845.

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

Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371

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.

Hanson MS, Hempel J, Brinton CC Jr. (1988) Purification of the Escherichia coli type 1 pilin and minor pilus proteins and partial characterization of the adhesin protein. J Bacteriol. 170(8), 3350–8.