Team:HUST-China/Demonstrate
「Demonstrate」
We believe our project-Rare Earth Enrichment By Optimized Tools(REEBOT) will achieve its goal for the following reasons:
Reference and theories
The whole circuit we build is based on the paper showed in JACS: Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions. In this paper we found that PmrA/PmrB two component system can be used as a trigger to start gene expression.
In this system, once ferric ion binds the exposed iron-binding motif in PmrB, the autophosphorylation of PmrB is activated, then comes phosphorylation of PmrA. And finally PmrA activates its own promoter (pmrC), which could lead to gene expression.
Circuits Construction
1. sensing part
In the sensing part, once the LBT sense lanthanide ions in the water, it will start the phosphorylation of PmrB and PmrA, eventually the PmrC is activated, the green fluorescent protein will also be translated.
Zichun Weng, Yating Hu, Qingyang Yu, Huiping Shi, Kangyuan Yu, Nan Hu, Shaofeng Liao,
If we could detect GFP, it means the sensing part could respond to lanthanide ions.
2. enrichment part and recycling part
In the capture part, firstly we use Sitag to combine our E.colis with silicon to finish recycling.
After that we combine GS-linker with LBTs. And in order to maximize cell potential, we triple this combination.
Once promoter T7 starts gene expression, protein oprF can be translated, which makes the GS-LBT be translated on the cell surface. In this way our E.coli can collect huge amounts of lanthanide ions. Thanks to flag-tag, we did immunoprotein detection and proved the feasibility of this circuit.
3.whole circuit
The whole circuit is as follows.
Modeling
At the beginning of the project, we release Reebot on the top of the container. While there are no lanthanide ions in environment, Reebot keep the initial status, and diffuse in the container. When 20 units of time pass, there are some lanthanide ions releasing at the bottom of the container. From the video, Reebot has a quick reaction and captures the lanthanide ions. After 100 units of time pass, other lanthanide ions release in another position at the bottom. Reebot successfully captures them too. Unfortunately, Reebot also meet waste problem in this situation. We may do some experiments using gradient method to confirm this problem and change the copy number or pathway to solve it.
It’s also the future plan of our project. Once we achieve it, we can release our Reebot in factory pool. If there is no lanthanide ion, Reebot lives as a normal E.coli. Once lanthanide ion emerges, Reebot can sense and capture it, then Reebot will combine with Si-board.
Experiments
Moreover, we find it is possible to build up the whole circuit due to our experimental results.
1.SDS-PAGE shows that the OprF-3* LBT(in this experiment we use OprF-3* LBT 4 as example), OprF-Sitag can be expressed on the surface of the e.coli.
2. Then we did immunofluorescence detection to figure out whether the cell surface display system works or not. According to Figure 3, the cell surface display of our LBTs is successful.
3. Then we used Precipitation titration detection for further verification by adding different concentration of terbium ions.
The amount of precipitation in WT group(with distilled water added) is much less than that in the test group. This huge difference in precipitation means capture part can recollect rare earth ions.
To be more convincing, we also did a precipitation-dissolving titration with hydrochloric acid.
Compared with control group, test group has significantly reduced the concentration of Tb3+ in the water. Our capture part really works.
