Section 1 : Introduction
Every system in REE Miner includes two devices: sensing and capture. The two devices in the system are related to each other through metabolic or regulatory pathways. Integrated with the systems，the E. coli BL21(DE3) can effectively sense specific ions in the environment and activates the expression of proteins that can capture the lanthanides. The protein of capture device will be firmly fixed on the surface of the cell membrane of the engineered bacteria and can be used with our hardware. We will prove the validity and feasibility from the following three aspects including construction, expression and application.
Section 2 : Construction
Each system consists of sensing and capture device which consists of several parts. The construction process is divided into two steps. First, build the sensing device and validate it . Then connect the capture device behind the sensing device.
In the initial stage of construction, we adapted seamless cloning of homologous recombination. Unique homologous sequences at front and rear ends of joining parts were designed based on Gibson assembly seamless cloning principle. After PCR, the parts underwent recombination reaction in the recombination enzyme system from some companies. However, due to the large number of fragments, the success rate of homologous recombination is low. In the end, we managed to build four systems. More details please click here.
Fig. 1. Pathways
Section 3 : Expression
The constructed systems were transformed into E. coli BL21(DE3) and expressed.
Chassis E. coli
PmrCAB[1] and GolS[2] systems both come from Salmonella bongori. They are more active in prokaryotes and can also work properly in E. coli BL21(DE3). E. coli BL21(DE3) can efficiently express foreign genes driven by T7 promoter. [3]It is beneficial to the purification of foreign proteins and suitable for the expression of non-toxic proteins.
PmrCAB and GolS systems
In order to express the genes at the right time, we introduced the PmrCAB and GolS systems. They've been engineered to specifically sense certain ions in the environment to activate the capture device.
Expression of parts
We first tested the sensing capabilities of the sensing device. Then SDS-PAGE was performed to verify the successful expression of parts that make up the sensing device. Finally, we verified the Pmr system by comparing the induced experimental group with the non-induced control group, and connected the GFP system after the GolS system to verify its normal function.Parts of sensing systems : PmrA, PmrB(LanM), PmrB(LBT-LanM), GolS(Cu2+).
As for the capture device, we designed several experiments to verify its various functions. Immunofluorescence labeling was used to verify OprF. Sitag was verified by silica adsorption experiment. The functions of Oprf-Sitag-LanM and Oprf-Sitag-dLBT-LanM were verified by REE adsorption experiments.
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Section 4 : Application
When we put the project into production, we not only need to make sure the theoretical of our path is correct, but also need a hardware to keep the engineered bacteria within certain limits. The engineered bacteria are stored and worked in the hardware, which prevents them from leaking into the environment. Our hardware boasts two special features: stratification and access-control. Through stratification, we are able to analyze the adsorption rate in different depths. 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. Finally, a sterilization chamber at the exit ensures that no engineered bacteria can escape into the environment. Click here for more details.
Fig. 2. Hardware structure
