Section 1 : Introduction

In 2022, we mainly worked on the recovery of REE from miner wastewater by using engineered microorganisms. Not only have we created two systems, but we also made several innovations in software and hardware. What’s more, we would also like to share our experience on the wet lab. We hope our project can give some assistance and guidance to the future iGEM teams.

Section 2 : Parts

The parts are essential to gene expression. Our project has registered multiple parts and four systems, all qualified for standardized requirement of RFC for the two system.

2.1 PmrCAB system

2.1.1 PmrA-PmrB(LanM)-pmrC promoter:

PmrB protein can phosphorylate PmrA protein once a lanthanide ion binds to PmrB(LanM), and the phosphorylated PmrA protein can activate the pmrC promoter, and then the target protein would be expressed.

2.1.2 PmrA-PmrB(FP)-pmrC promoter:

PmrB protein can phosphorylate PmrA protein once a lanthanide ion binds to PmrB(FP), and the phosphorylated PmrA protein can activate the pmrC promoter, and then the target protein would be expressed.

2.2 GolS system:

GolS（Cu²⁺）-PgolB:

The GolS protein can active the PgolB once a Copper ion and then the target protein would be expressed.

2.3 Oprf-Si-tag-dLBT-LanM:

The Oprf protein can act as an anchored protein on E. coil’s surface, with which, LanM, a lanthanide-binding tag can capture lanthanide ions around the bacteria.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.

Section 3 : Experience of wet lab

In 2017, we developed a system with PmrB(LBT) to recover REE, which works with relatively low efficiency. Therefore, in 2022, we replace LBT with LanM and dLBT-LanM, which reflect higher efficiency and can collect more kinds of REE. What’s more, we designed a new system, GolS system. The GolS system is activated by Au³⁺ in nature, and we changed its extracellular proteins, so that the new GolS system can be activated by Cu²⁺, which is common in wastewater, thus we are able to collect more REE.

3.1 genetic engineering:

In the original design, we have added corresponding homology arms to each fragment, so the parts can be connected easily.

During our first cycle of experiment, we tried to connect the parts and the carrier by Homologous Recombination. Nonetheless, we failed in building the PmrCAB system after several tries. Considering that the part number in PmrB is larger, which may lead to a lower possibility of connecting correctly, we decided to overlap some small parts first. We used SOE PCR to first connect PmrB to pmrC promoter, then PmrB-pmrC promoter to oprf-sitag-LanM. And finally we successfully constructed the PmrCAB system.

3.2 engineering bacteria:

In the process of expression, we used Oprf, an anchoring protein, to fix our complex proteins on the outside of the cell membrane to capture the REE in wastewater. In addition, we added His-tag to the C-end of the Oprf to identify whether the protein was successfully anchored to the cell surface. And we added His-tag to the end of the PmrA gene sequence for subsequent protein purification and identification. What’s more, in order to enhance the expression effect, we chose the potent promoter T7 to initiate the expression of protein PmrA, PmrB, thereby enhancing the expression efficiency of the Pmr two-component system.

From the picture taken in dark, we can draw the conclusion that the proteins have expressed successfully from the Green fluorescence. And through comparing figure A and B, we can tell that most GolS engineering bacteria contain successfully expressed proteins.

The red line shows the OD₆₀₀ of engineering bacteria uninduced and the black line shows the OD₆₀₀ engineering bacteria induced. The induced engineering bacteria reflects fairly good adsorption rate.

Section 4 : Hardware

Being inspired by the distillation tower, we built our hardware, a layered long barrel structure with each layer as a separator. And we add a sterilization chamber at the exit. The barrel is filled with silicon dioxide and our engineering bacteria to better recover REE. When the miner wastewater flows into the column with the buttons closed, the REE can be captured, and after several hours, we can open the buttons to fetch water lacking REE, leaving the REE in the column.