Difference between revisions of "Part:BBa K3610015"

Latest revision as of 03:22, 28 October 2020

LargeBit NanoLuc - codon optimized for C. reinhardtii

This part is a modified version of Part:BBa_K1761005. It has been codon optimized for the expression in C. reinhardtii.

Usage and Biology

NanoLuc is a small, bright and stable luciferase from O. gracilirostris and has been proven to be an efficient reporter protein since its introduction by Promega. One way to use the NanoLuc luciferase is in a protein-fragement complementation assay (PCA) to visualize protein-protein or protein-ligand interaction. The protein-protein interaction is observed by fusing spli-reporter proteins, like the LargeBit and the SmallBit from NanoLuc, to the proteins of interest. Interaction of the two proteins will associate the reporter fragments and lead to a reconstitution of the full length and fully functional NanoLuc protein.

To enhance translation, we codon optimized this part for the expression in C. reinhardtii as codon bias has shown to influence mRNA stability and translational efficiacy. C. reinhardtii has a strong codon bias and additionally a high GC content, oftentimes making efficient expression of foreign sequences in C. reinahrdtii very difficult.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3610015 short</partinfo>
-This part is a modified version of [[Part:BBa_K3168002]]. It has been codon optimized for the expression in C. reinhardtii.
+This part is a modified version of [[Part:BBa_K1761005]]. It has been codon optimized for the expression in C. reinhardtii.
 ===Usage and Biology===
 NanoLuc is a small, bright and stable luciferase from O. gracilirostris and has been proven to be an efficient reporter protein since its introduction by Promega. One way to use the NanoLuc luciferase is in a protein-fragement complementation assay (PCA) to visualize protein-protein or protein-ligand interaction. The protein-protein interaction is observed by fusing spli-reporter proteins, like the LargeBit and the SmallBit from NanoLuc, to the proteins of interest. Interaction of the two proteins will associate the reporter fragments and lead to a reconstitution of the full length and fully functional NanoLuc protein.
-To enhance translation, we codon optimized this part for the expression in S. cerevisiae as codon bias has shown to influence mRNA stability and translational efficiacy.
+To enhance translation, we codon optimized this part for the expression in C. reinhardtii as codon bias has shown to influence mRNA stability and translational efficiacy. C. reinhardtii has a strong codon bias and additionally a high GC content, oftentimes making efficient expression of foreign sequences in C. reinahrdtii very difficult.
-For our project we use this part for better characterization of the split NanuLuc system by making experiments with FRB and FRKP. We further used it for our biosensing system which relies on the dimerization of plant pathogen recognition receptors (PRRs). The general idea is that a microbe assiciated molecular pattern (MAMP), like the epitope flg22 of flagellin, binds to a cell surface receptor, which drives the interaction of the receptor with a coreceptor. The split luciferase proteins are fused to the N-terminal intracellular domains of the receptor protein.