Latest revision as of 14:46, 12 October 2022

PbrR -> C. metallidurans

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. The binding capacity may be concerned with three conserved Cys residues: Cys79, Cys114 and Cys123. 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.

Usage and Biology

This part encodes for PbrR, a metal binding protein that can bind with heavy metal ions such as lead and cadmium highly specifically. The binding affinity of it is shown as follows.

Figure 1. PbrR protein. (Left) Structure of OmpA-PbrR fusion protein; (right) Metal-binding specificity of PbrR

Characterisation of PbrR

Protein PbrR is a special metal protein found in Cupriavidus metallidurans that specifically binds to Pb2+ ions. In realistic research we further examined that this protein also bears a high affinity to Cd2+ ions(Figure 1). We demonstrated in our project that when we fused PbrR with OmpA, the binding of PbrR with Cd2+ is greatly enhanced.

Figure 2. Photocatalytic reduction of MV by TiO2 and induced CdS nanoparticles

For organism M.thermoacetica, this kind of bacteria can produce S2- ions from cysteine and forms a higher sulfur concentration around the cell which then induces the precipitation of CdS nanoparticles when Cd2+ ions are added into the media. We assume that if we form a same local high concentration of Cd2+ with fused protein OmpA-PbrR on the outer cell membrane, we can also achieve a similar precipitation of CdS nanoparticles on to the walls of E.coli, the well model bacteria. To confirm the capability of our CdS system based on OmpA-PbrR, we conducted the same photo-catalytic assay. Bacteria were divided into three groups. Bacteria were induced to express OmpA-PbrR protein and cultured with both Cd2+ and S2- in the experiment group. Groups that either lacked induced expression or necessary ions to build semiconductors were negative controls. We found that illumination resulted in a same increasing trend in experiment group (Figure 2). This confirmed the photo-catalytic capability of our PbrR-based precipitation of semiconductors.

Figure 3. TEM image of native E.coli cells (right) and cells with in situ formed CdS nanoparticles (left)

We also did a TEM imaging of the CdS particles formed on bacteria surface and demonstrated that the CdS particles are nanoparticles. To conclude, we successfully expressed PbrR onto the surface of E.coli and proved its heavy metal binding specificity. It can adsorb cadmium ions with high affinity. When we add sulfide ions into the solution, CdS nanoparticles can be fixed on the cell surface and conduct electrons into the cells.