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.
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.
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.
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.
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