Section 1 : Introduction
We have designed two expression systems to realize the function of capturing rare earth element ions. Each system has a sensing part and a capture part. When the sensing part perceive target ions, the capture part will be activated and act as a miner to adsorb them.
Section 2 : PmrCAB system
We noticed a two-component and phosphorelay signal transduction system called PmrA/PmrB in Salmonella bongori, which is a sort of lanthanide-utilizing bacterium. PmrA is an intracellular protein, which could activate pmrC promoter. PmrB is a membrane-anchored protein, which originally has an iron(III)-binding motif fixed outside the cell membrane, which allows it to sense ferric ions in the surroundings. We modified the motif into PmrB (LanM) and PmrB (LBT-LanM). The domain of LBT could sense Tb3+ in the solution while the domain of LanM could sense a wider range of lanthanide ions. After modified motif sensing target ions, PmrB would phosphorylate PmrA, which will then activate the pmrC promoter. Consequently, genes under the sequence could be driven to be expressed [1].
Section 3 : GolS system
We also designed another expression system which is also found in Salmonella bongori, GolS system. Protein gols is sensitive to Au3+ and Cu+ in the environment. These two element ions do not commonly occur in drainage samples, so we modified a sequence of amino acids in the protein, as a result of which, it could sense Cu2+ in the surroundings [2]. Cu2+ in drainage would be detected by modified protein. Upon sensing these ions, the downstream promoter is activated and genes could be transcribed. As a result, the fusion protein could be expressed [3].
Section 4 : Fusion protein
We designed two types of fusion protein acting as the capture part. The fusion protein is composed of Oprf, Si-tag, LanM and the linkers between them, while we also tried replacing LanM with LanM-dLBT. Oprf is a membrane-anchored protein, which could locate the fusion protein on the cell membrane. GS linkers are placed between Oprf, Si-tag and LanM, while we used rigid linkers to connect dLBT and LanM. Si-tag binds specifically and reversibly to the silica material so the engineered bacteria cells would be located on the silicon column, so it could be used for locating functional proteins on silica surfaces [4-5]. LanM has four metal-binding EF hands motifs, resulting in the high affinity to rare earth elements [6]. Lanthanide-binding tag (LBT) has been proved to be able to selectively bind to lanthanide ions with high affinity and have been applied to capture lanthanide ions in some research [7-8]. Double-Lanthanide-binding tag (dLBT) could bind to two lanthanide ions at the same time, and we linked dLBT with LanM in order to enhance the capacity of the fusion protein to capture the target ions. As a result, two kinds of fusion proteins: Oprf-Sitag-LanM and Oprf-Sitag-dLBT-LanM would be regulated by PmrCAB or GolsS system, forming four pathways.
Section 5 : Hardware
We designed a silicon adsorption column to effectively adsorb the Si-tag along with the engineering bacterium and the rare-earth element. Compared with common adsorption columns, our column boasts two special features: stratification and access-control. Through stratification, we are able to analyze the adsorption rate in different depths. We can also decide the amount of quartz sand we add through the stratification strategy. On the other hand, with the help of access-control, we could store the wastewater for as much time as we want to. This helps the silicon combine more bacteria, and at the same time, more rare-earth elements. On the whole, the silicon adsorption column helps us conduct our experiment more effectively and precisely.
