Difference between revisions of "Part:BBa K3610048"

Latest revision as of 01:08, 28 October 2020

CORE ectodomain / mCherry C-terminal for S. cerevisiae

This part entails the ectodomain of the plant pattern recognition receptor CORE from S. lycopersicum which is fused to the C-terminal domain of the mCherry protein via a 15 amino acid linker. The C-terminal sequence of the receptor protein, encoding the signal peptide, was removed from the sequence. To ensure localization, the secretion signal of the alpha Factor from yeast is added instead.

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 mCherry

In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the C-terminal domain of the split mCherry 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. It is possible that dimerization of the two receptors can be induced without the intracellular kinase domain of neither CORE nor BAK1. We were interested in testing whether coexpression of this part with BAK1 fused to the N-terminal domain of split-mCherry in S. cerevisiae would allow to visually capture the presence of the bacterial epitope csp22 in water samples by ligand-dependent interaction of the two receptors, an interaction which would drive the reconstitution of the split-mCherry protein.

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_K3610048 short</partinfo>
-This part entails the ectodomain of the plant pattern recognition receptor CORE from A. thaliana which is fused to the C-terminal domain of the mCherry protein via a 15 amino acid linker. The C-terminal sequence of the receptor protein, encoding the signal peptide, was removed from the sequence. To ensure localization, the secretion signal of the alpha Factor from yeast is added instead.
+This part entails the ectodomain of the plant pattern recognition receptor CORE from <i>S. lycopersicum</i> which is fused to the C-terminal domain of the mCherry protein via a 15 amino acid linker. The C-terminal sequence of the receptor protein, encoding the signal peptide, was removed from the sequence. To ensure localization, the secretion signal of the alpha Factor from yeast is added instead.
 ===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.
+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 <i>S. lycopersicum</i> has been confirmed by expressing the receptor in <i>A. thaliana</i>, which made the plant responsive to csp22, a PAMP that is otherwise not perceived by PRRs from <i>A. thaliana</i>.
@@ Line 13: / Line 13: @@
 In this case, the C-terminal domain of CORE, entailing the intracellular kinase domain, was removed from the sequence. Instead, the C-terminal domain of the split mCherry 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 [[Part:BBa_K3610034]], which is the ectodomain of BAK1 fused to the N-terminal part of mCherry, csp22-induced interaction between BAK1 and EFR drives the reassembly of the C-terminal and N-terminal domain of the split-mCherry, reconstituting its functionality as a fluorescent protein. This part, therefore, allows visualization of the ligand-dependent interaction of the plant PRRs EFR and BAK1.
+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. It is possible that dimerization of the two receptors can be induced without the intracellular kinase domain of neither CORE nor BAK1. We were interested in testing whether coexpression of this part with BAK1 fused to the N-terminal domain of split-mCherry in <i>S. cerevisiae</i> would allow to visually capture the presence of the bacterial epitope csp22 in water samples by ligand-dependent interaction of the two receptors, an interaction which would drive the reconstitution of the split-mCherry protein.
-This enables us to use this part, in coordination with [[Part:BBa_K3610034]], to visually capture the presence of the csp22 epitope in water samples as the csp22 pattern will induce interaciton between the receptors, causing the split-mCherry parts to rejoin and generate a funcitonal fluorescent protein.
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