Difference between revisions of "Part:BBa K3945009"

Latest revision as of 00:17, 22 October 2021

Lucifer: A luminescence-based measurement system for rare earth elements

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 measuring protein-protein interactions [1]. The substrate required for the NanoBIT systems, and other systems that use NanoLuc is furimazine, which is a coelenterazine analog. Upon the NanoBiT proteins interacting with one another, this substrate will be chemically converted such that a light signal will be emitted, which can be measured by a luminometer. Furimazine is primarily sold in Promega’s Live Cell Assay and other similar kits.

We have combined the NanoBIT technology with the lanmodulin to create a selective rare earth element 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.

Design

When designing the Lucifer construct, it was extremely important to note that the two luciferase subunits, Large BiT and Small BiT, were as close to each other as possible. As mentioned previously, unless these subunits come into very close proximity, the likelihood of signal output is low [1]. As such, the best linker sequence selected was done through protein modelling experiments, where the linkers were tested through homology modelling to determine the atomic distance between the NanoBiT subunits in the desired fusion protein. To further test this, molecular docking simulations were performed to ensure LanM still retains functionality even with the added protein.

Experimental Plan

In order to measure REE concentration using the Lucifer system, there were several steps we would need to undergo. After successful protein production, we would need to determine background signal emittance. We would do this by measuring the luminescence output of just the protein and the added furimazine. Afterwards, a calibration curve using known REE concentrations will have to be calculated. This will be done by resuspending a constant amount of the protein in differing concentrations of REE solution, adding furimazine, and measuring the luminescent output using a luminometer. Once this calibration curve was completed, we would test the REE solution after it had already undergone the metal separation step of our system. This would help us determine the range of detection of Lucifer, and also determine the viability of it for testing REE concentrations after the Neocycle separation process. This experimental process will be identical to the BRET experimental process apart from the wavelength at which the signal output is measured. Future optimization experiments will be conducted to better understand the range of the Lucifer system.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 586
1000
COMPATIBLE WITH RFC[1000]

References

1. Promega. 2021. NanoBiT PPI Starter Systems. Retrieved online from https://www.promega.ca/products/protein-interactions/live-cell-protein-interactions/nanobit-ppi-starter-systems/?catNum=N2014

@@ Line 13: / Line 13: @@
 ===Design===
 <p>When designing the Lucifer construct, it was extremely important to note that the two luciferase subunits, Large BiT and Small BiT, were as close to each other as possible. As mentioned previously, unless these subunits come into very close proximity, the likelihood of signal output is low [1]. As such, the best linker sequence selected was done through protein modelling experiments, where the linkers were tested through homology modelling to determine the atomic distance between the NanoBiT subunits in the desired fusion protein. To further test this, molecular docking simulations were performed to ensure LanM still retains functionality even with the added protein.</p>
+===Experimental Plan===
+<p>In order to measure REE concentration using the Lucifer system, there were several steps we would need to undergo. After successful protein production, we would need to determine background signal emittance. We would do this by measuring the luminescence output of just the protein and the added furimazine. Afterwards, a calibration curve using known REE concentrations will have to be calculated. This will be done by resuspending a constant amount of the protein in differing concentrations of REE solution, adding furimazine, and measuring the luminescent output using a luminometer. Once this calibration curve was completed, we would test the REE solution after it had already undergone the metal separation step of our system. This would help us determine the range of detection of Lucifer, and also determine the viability of it for testing REE concentrations after the Neocycle separation process. This experimental process will be identical to the BRET experimental process apart from the wavelength at which the signal output is measured. Future optimization experiments will be conducted to better understand the range of the Lucifer system. </p>
 ===Sequence and Features===