Difference between revisions of "Part:BBa K3945009"
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===Design=== | ===Design=== | ||
| − | <p> | + | <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> |
===Sequence and Features=== | ===Sequence and Features=== | ||
Revision as of 00:11, 22 October 2021
Lucifer: A luminescence-based measurement system for rare earth elements
Usage and Biology
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 586
- 1000COMPATIBLE 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