Difference between revisions of "Part:BBa K3945009"
Line 8: Line 8:
 
===Usage and Biology===
 
===Usage and Biology===
 
<p>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 <i>Oplophorus gracilirostris</i>, 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. </p>
 
<p>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 <i>Oplophorus gracilirostris</i>, 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. </p>
−
<p>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.</p>
+
<p>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.</p>
  
−
<p>A novel fusion protein with lanmodulin fused to the N-terminus of <I> Clostridium thermocellum’s </i> 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 [3]. </p>
 
  
 
===Design===
 
===Design===
Line 21: Line 20:
  
 
<p>  
 
<p>  
−
1. JA C, ER F, JA M, JV H, TN L. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. Journal of the American Chemical Society. 2018 [accessed 2021 Sep 17];140(44):15056–15061. https://pubmed.ncbi.nlm.nih.gov/30351021/. doi:10.1021/JACS.8B09842 </p>
+
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 </p>
−
 
+
−
<p> 2. GJ D, JA M, DM P, DW R, JA C, Y J. Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin. Inorganic chemistry. 2020 [accessed 2021 Sep 17];59(17):11855–11867. https://pubmed.ncbi.nlm.nih.gov/32686425/. doi:10.1021/ACS.INORGCHEM.0C01303
+
−
</p>
+
−
<p> 3.Improved immobilization of fusion proteins via cellulose‐binding domains - Linder - 1998 - Biotechnology and Bioengineering - Wiley Online Library. [accessed 2021 Oct 14]. https://onlinelibrary.wiley.com/doi/pdf/10.1002/(SICI)1097-0290(19981205)60:5%3C642::AID-BIT15%3E3.0.CO;2-8 </p>
+

Revision as of 00:09, 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

With a flexible and inert Threonine-Proline linker joining the two proteins, the immobilized lanmodulin will be able to operate freely on the surface of the cellulose support. The sequence of both proteins was also codon-optimized for expression in E. coli. A Factor Xa recognition site has been incorporated between the CBM and lanmodulin to cleave the lanmodulin protein free after purification on cellulose

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