Efficient Recovery

Electronic waste has proved to be a valuable source of industrially important rare earth elements, however recycling of rare earth elements from this waste stream has been due to lack of cost-effective metal recovery technologies. To tackle this issue, we have contributed three molecular biology tools featuring lanmodulin, a highly selective lanthanide binding protein. These parts for allow for efficient purification and immobilization of lanmodulin for use in in a high throughput system of rare earth element recovery.

LanM-His (BBa_K3945001)

Lanmodulin is recently a novel lanthanide binding protein that displays more than 100-million fold selectivity for rare earth elements1. Such affinity and selectively has not been observed in any previously studied macromolecule. In addition, lanmodulin is extremely robust, capable of withstanding temperatures as high as 95 °C and pH levels as low as 2.52. Thus, allowing it be the perfect molecular tool for us in an efficient rare earth recovery system. The lanmodulin sequence has been codon optimized for expression in E. coli and with the addition of a 6x histidine tag to C-terminus of the protein, it could easily be purified using the universal His-Tag purification protocol.

LanM-Cex (BBa_K3945007)

A novel fusion protein with lanmodulin fused to the N-terminus of Cellulomonas fimi’s Cex cellulose binding module CBMcex. CBMcex has strong affinity for a variety of cellulose type, especially microcrystalline cellulose over a wide variety of conditions and pH ranges. Thus, allowing for efficient one-step immobilization and purification of lanmodulin onto a cellulose support. With a flexible and inert Threonine-Proline linker joining the two proteins, the immobilized lanmodulin will be able to operate freely on surface of the cellulose support.

LanM-CipA (BBa_K3945008)

A novel fusion protein with lanmodulin fused to the N-terminus of Clostridium thermocellum’s CipA cellulose binding module (CBMcipa). CBMcipa is a versatile and is able to from stable connections with both crystalline and regenerated amorphous cellulose. In addition, CBMcipa is one of the most characterized CBMs out there and has been shown to have a high efficiency of heterologous expression in E. coli, making an ideal choice for one step immobilization and purification of LanM from E. coli onto cellulose solid support.

Selective Measurement

Quantification of trace elements is often a difficult task which required access to expensive technologies such as inductively coupled plasma mass spectrometry (ICP-MS). This is particularly the case with REEs as they are often found in low concentrations in either metal ores or electronic waste. As such we have harnessed the power of lanmodulin to develop three versatile molecular tools for selective measurement and detection of REEs. The three systems are designed to produce either a luminescence, fluorescence or electrochemical signal, making them applicable to a wider variety of conditions and applications.

Lucifer (BBa_K3945009)

The NanoLuc Binary Technology, or NanoBIT for short, is a patented split luciferase complementation system from Promega (the system is composed of a Large Bit (LgBiT) and Small Bit (SmBiT), which upon binding to each other, emit a luminescent signal [1]. Unlike other luciferase systems, which are typically based off of firefly luciferase, NanoBiT is much smaller in size to its derivation from Oplophorus gracilirostris, a deep sea shrimp [1]. In order to ensure that the background noise for this system is low, the proteins have very low binding affinity to each other, meaning they won’t bind unless in very close proximity. As such, this system is ideal for construction of an efficient biosensor. We have combined the NanoBIT technology with the lanmodulin to create a selective REE measurement construct. In this system the LgBiT and SmBiT are fused to opposite ends of lanmodulin. Upon binding to REEs, lanmodulin folds into its secondary structure bringing to luciferase fragments together which will produce a quantifiable luminescence signal.

BRET (BBa_K3945010)

The BRET system utilizes mCherry as a red fluorescent protein derived from Discosoma sea anemones, well-known for its use in molecular biology as well as the NanoLuc luciferase enzyme from Promega, derived from Oplophorus gracilirostris [2]. The two proteins are added to the opposite ends of the lanmodulin. When lanmodulin binds REEs, it brings the mCherry and NanoLuc together. These two proteins undergo the phenomena of BRET (bioluminescence resonance energy transfer), which is a non-radiative energy transfer between a luminophore donor (NanoLuc) and a fluorophore acceptor (mCherry) [3]. Upon this transfer of energy, a fluorescence signal is produced in the 550-650 nm which could be measure using a fluorometer. As such, this system can be used to determine whether LanM is binding to lanthanide ions, and the intensity of the signal can quantify the ion concentration.

Elektra (BBa_K3945011)

The Elektra system is compromised of the lanmodulin protein with addition of a couple of key amino acids on its terminal ends. The cysteine amino acid on the C-terminal end will be used to covalently couple lanmodulin to a gold electrode via EDC coupling. On the N-terminal end of lanmodulin there is a histidine residue which will allow a ruthenium signal molecule to be attached onto the other end of lanmodulin to create Elektra. Upon binding to lanthanides the N-terminal end of lanmodulin will be brought closer to the gold electrode and at the certain voltage, the ruthenium molecule redox potential will change, emitting an electrochemical signal that can be measured using a potentiostat. As such Elektra can quickly transform protein metal binding to a measurable electrochemical signal.

Improved Lanmodulin

Lanmodulin already portrays tremendous ability to selectively bind rare earth element with three high affinity binding pockets. The fourth pocket of lanmodulin however has considerably lower affinity for REE compared to the other binding pockets. Due to the novelty of the protein no work has previously been done to optimize the fourth binding pocket We put powers of modelling and computational analysis together to create 5 lanmodulin mutants with predicted improved functionality of the fourth binding pocket.

Figure 1. Summary of the mutant lanmodulin proteins and the methods/algorithm used to generate them.

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Team:Calgary/Part Collection