Difference between revisions of "Part:BBa K3610051"

Revision as of 22:11, 16 October 2020

CORE ectodomain / SmallBit NanoLuc for S. cerevisiae

This part includes the ectodomain of the plant pattern recognition receptor CORE fused to the SmallBit part of the split-NanoLuc system. To ensure localization at the membrane, this part further contains the sequence for the signal peptide of the alpha factor from S. cerevisiae.

Usage and Biology

CORE

The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there additionally is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.

Usage with NanoLuc

In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.

The ligand-dependent interaction of CORE with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither CORE nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, csp22-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs CORE and BAK1. This enables us to use this part, in coordination with the BAK1 ectodomain and LargeBit NanoLuc, to visually capture the presence of the csp22 epitope in water samples, as the csp22 pattern will induce interaction between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a functional protein, which gives a visual uotput with the substrate furimazine.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1740
Illegal BamHI site found at 373
Illegal BamHI site found at 1765
Illegal BamHI site found at 2117
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3610051 short</partinfo>
-This part includes the ectodomain of the plant pattern recognition receptor CORE fused to the SmallBit part of the split-NanoLuc luciferase. The sequence for the receptor and the Luciferase protein have been codon optimized for expression in C. reinhardtii. To ensure localization at the membrane, this part further contains the sequence for the signal peptide SP7 from C. reinhardtii and the self-cleaving protein from the foot and mouth virus.
+This part includes the ectodomain of the plant pattern recognition receptor CORE fused to the SmallBit part of the split-NanoLuc system. To ensure localization at the membrane, this part further contains the sequence for the signal peptide of the alpha factor from S. cerevisiae.
 ===Usage and Biology===
-====CORE====The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there additionally is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.
+====CORE====
+The cold shock protein receptor (CORE) is a plant pattern recognition receptor (PRR) and as such activates host innate immunity through detection of pathogen-associated molecular patterns (PAMPs). CORE is a leucine-rich repeat receptor-like kinase with 22 LRRs, there additionally is a 6 amino acid insert at LRR 11. It consists of an extracellular domain that perceives an epitope, csp22, from the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), which are highly abundant proteins found in the cytosol of bacteria. Further domains are a single pass transmembrane domain and an intracellular kinase domain (The sequence encoding the kinase domain is not in this part). Interaction of CORE with brassinosteroid-associated kinase (BAK)1 is necessary for inducing an immune response in the plant. The dimerization of CORE and BAK1 depends on the csp22, the ligand of CORE. The function of CORE in S. lycopersicum has been confirmed by expressing the receptor in A. thaliana, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from A. thaliana.
-In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the N-terminal domain of the split mCherry was fused to the C-terminal domain via a 15 amino acid linker.
+====Usage with NanoLuc====
+In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the SmallBit part of the split NanoLuc luciferase was fused to the C-terminal domain via a 15 amino acid linker.
-This part is used in the same way as Part:BBa_K3610047, meaning as a way to visualize the presence of the csp22 protein together with the coreceptor. It is, however, modified for expression in C. reinhardtii instead of S. cerevisiae.
+The ligand-dependent interaction of CORE with its coreceptor BAK1 is driven by the extracellular ligand-binding domain. Further necessary is the transmembrane domain, including the juxtamembrane domain. Therefore, dimerization can be achieved without the intracellular kinase domain of neither CORE nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the LargeBit part of the NanoLuc luciferase, csp22-induced interaction between BAK1 and EFR can drive the reassembly of both parts from the NanoLuc luciferase, reconstituting its function to react with furimazine in the presence of oxigen, yielding furimamide and a fluorescent output. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs CORE and BAK1. This enables us to use this part, in coordination with the BAK1 ectodomain and LargeBit NanoLuc, to visually capture the presence of the csp22 epitope in water samples, as the csp22 pattern will induce interaction between the receptors, causing the split-NanoLuc luciferase parts to rejoin and generate a functional protein, which gives a visual uotput with the substrate furimazine.
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