Team:HUST-China/Description
「Description」
Rare earth elements, especially how to capture them, have attracted numerous scholars to explore. They have successfully developed some functional sensors for some rare earth elements. However, the lanthanide elements, one of the important and wealthy rare earth elements, are not included, so our team plans to follow some researches in this field.
We have designed an intelligent system, which would not react unless the concentration of lanthanide ions reaches the threshold.
To reach our goal, we need a smart sensing circuit. Fortunately, we found a two-component system called PmrA/PmrB aimed at detecting ferric ions. If we can find some appropriate sequences encoding particular proteins to adsorb lanthanide ions, we can replace the iron-binding sequence with them to achieve our goal.
Consulting a great amount of literatures, we made it! And we learned that the most efficient lanthanide binder is lanthanide-binding tag, abbreviated as LBT. LBT contains a domain that can capture lanthanide ions, such as Tb ions. In other words, lanthanide ions can specifically bind to LBT. Exploring lots of literatures, we found 12 kinds of LBTs, attempting to measure their abilities to capture lanthanide ions. 12 kinds of LBTs differ from each other in sequences and structures, which suggests that their adsorptions of lanthanide ions are different.
The sensing part is based on a 2-component system called PmrA /PmrB system. PmrA encodes an intracellular protein, and PmrB encodes a membrane-anchored protein, while PmrC is a promoter. The original PmrB protein contains a domain called ferric binding tag. In molecular construction, we replace that domain with 12 kinds of LBT, our key protein.Once a Tb ion, one of the lanthanide ions binds to LBT, PmrB protein would change its conformation, so it would phosphorylate PmrA protein. And then, the phosphorylated PmrA protein would activate PmrC promoter, initiating the expression of downstream genes. As a result, GFP would be lightened, reporting that the sensing section senses a lanthanide ion. And that’s how the sensing section works.
Ions sensing is just the first step, then we need to capture them. In this part, we also use LBT——our lanthanide hunter.
To amplify the enrichment of lanthanide ions, we design a triple LBTs, so that it can capture much more lanthanide ions. Oprf is a membrane anchored protein, and flag is used for immunofluorescence assay. With the help of oprf, the recombinant protein, oprf-flag-triple LBTs would be displayed on the outer surface of the cell membrane, so it can capture much more lanthanide ions surrounding to reach the goal of enrichment.
Now our bacteria have captured a lot of lanthanide ions. What we need to do at last is just collecting our cells. Referring to the project of 2015 iGEM HUST-China, we redesign a circuit including sitag, which has been proved to be functional in eukaryotic circuit. Sitag is a protein that can bind to a silica board, and thus, our bacteria would be attached to that silica board, and then be recycled.
The circuit below can illustrate our project better:
To meet our goal that developing an intelligent and efficient system, the complete design of REEBOT is a perfect combination of the sensing system and the capture system. With the existence of arabinose, PmrA and PmrB(LBT) proteins would be expressed conducted by araBAD promoter. The system is put to sleep if no lanthanide ions surrounding, but once a lanthanide ion bind to LBT, PmrB(LBT) would phosphorylate PmrA, and then the phosphorylated PmrA would activate PmrC promoter. As a result, 3 LBTs, anchored on the outer surface of the cell membrane, would be expressed to amplify the capture reaction. In the same time, the Si-tag would be expressed, enabling the bacteria to be adsorbed to a silica net. The 3 sections are tightly connected. The sensing section is like a switch. Tb ions existing, switch on; Tb ions absent, switch off. The enrichment section and the recycling section are conservatively controlled by the sensing system. The sensing section would not be awakened unless a Tb ion binds to LBT, and the enrichment section and the recycling section would not be activated unless the sensing section is awakened. To sum up, our engineering bacteria can intelligently and efficiently sense and then capture much more lanthanide ions, and finally recycled by a silica board.
