PbrR is amplified from Cupriavidus metallidurans CH34 (formerly Ralstonia metallidurans) genome.
PbrR, together with its homologues in the same bacterium, are the only known lead(II)-specific binding protein found in nature. PbrR binds lead(II) 1000-fold more selectively over other metal ions such as mercury(II), zinc(II), copper(II), nickel(II), and silver(I). Besides lead(II), PbrR also shows the binding capacity of cadmium(II), but slightly lower than that of lead(II). The binding capacity may be concerned with three conserved Cys residues: Cys78, Cys113 and Cys122.
Because the gene encoding PbrR contains a digest site of Pst I (-CTGCAG-), to fit the construction of standard biobrick, we change those site from -CTGCAG- to -CTGCAA- by point mutation.
The source of this part: Cupriavidus metallidurans CH34
Our project is to construct an artificial solar-driven system in model organisms. There are two main problems to be solved when we artificially construct solar-driven systems: media for electron transportation and enzymes to harvest electrons to chemical bonds. We successfully solved the two problems in E.coli and testes the efficiency of our system using hydrogen production.
To solve the problem of electron transduction, we fused PbrR (BBa_K1958007), which is another basic part of our project this year, with a previously existing basic part OmpA (BBa_J36836). PbrR is a kind of metal binding protein which can adsorb cadmium ions. OmpA is one of the outer membrane protein of E.coli. By fusing these two proteins together, we can construct a Biobrick device that can display PbrR onto the surface of E.coli so that cadmium ions can be fixed! When we add S2- into the culture medium, CdS can be fixed firmly onto the surface of E.coli and conduct electron transportation into the bacteria. The detailed experiment results are listed as follows.
The PbrR (BBa_K1958007) based artificial PS system is constructed according to our design. The first step to verify an artificial PS system is to test its photocatalytic capability. The redox dye methyl viologen (MV) is a well-establish indicator. In a photocatalytic reaction, photons spark excited electron on the semiconductor which then reduce MV from its +2 oxidized state to +1 reduced state (figure 1). Increased concentration of reduce MV leads to an increase of absorbance at 605nm.
