Difference between revisions of "Part:BBa K3610041"

Latest revision as of 00:24, 28 October 2020

EFR ectodomain / mCherry N-terminal for C. reinhardtii

This part includes the ectodomain of the plant pattern recognition receptor EFR from A. thaliana fused to the N-terminal domain of the split-mCherry protein. The sequence for the receptor and the mCherry 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 disease (FMD) virus.

Usage and Biology

EFR

Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.

Usage with mCherry

In this case, the C-terminal domain of EFR, 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.

The ligand-dependent interaction of EFR 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 EFR nor BAK1.
We were interested in testing whether coexpression of this part with BAK1 fused to the C-terminal domain of split-mCherry in C. reinhardtii would allow to visually capture the presence of the bacterial epitope elf18 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
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 93
Illegal NheI site found at 235
Illegal NheI site found at 1165
Illegal NheI site found at 2083
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 148
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1751
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3610041 short</partinfo>
-This part includes the ectodomain of the plant pattern recognition receptor EFR fused to the N-terminal domain of the split-mCherry protein. The sequence for the receptor and the mCherry 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 EFR from <i>A. thaliana</i> fused to the N-terminal domain of the split-mCherry protein. The sequence for the receptor and the mCherry protein have been codon optimized for expression in <i>C. reinhardtii</i>. To ensure localization at the membrane, this part further contains the sequence for the signal peptide SP7 from <i>C. reinhardtii</i> and the self-cleaving protein from the foot-and-mouth disease (FMD) virus.
 ===Usage and Biology===
 ====EFR====
-Elongation factor-thermo unstable receptor (EFR) from A. thaliana is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucin-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucin-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.
+Elongation factor-thermo unstable receptor (EFR) from <i>A. thaliana</i> is a plant pattern-recognition receptor (PRR). It is a cell surface receptor and part of the plants firts defence mechanism against potential pathogens. The EFR receptor is also a leucine-rich-repeats (LRR) receptor-like serine/threonine-protein kinase. The protein consists of an extracellular domain with leucine-rich repeats, a ligand binding domain found in many receptors, a single-pass transmembrane domain and finally an intracellular kinase domain. The ligand binding domain from EFR has high specificity to a bacterial pathogen-associated moleculat pattern (PAMP), namely the epitope elf18 of the abundant protein Elongation Factor Tu (EF-Tu), which is catalyzes the binding of aminoacyl-tRNA (aa-tRNA) to the ribosome in most prokaryotes and therefore is evolutionarily highly conserved. This makes the EFR a receptor that can be activated by the presence of a huge variety of bacteria. Upon binding of the ligand to the extracellular domain, the receptor dimerizes with its coreceptor BRI1-associated receptor kinase (BAK1). This interaction triggers the activation of the intracellular kinase domain of EFR and BAK1, initiating a signal cascade leading to an upregulation of immune response mechanisms.
 ====Usage with mCherry====
@@ Line 13: / Line 13: @@
 In this case, the C-terminal domain of EFR, 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.
-The ligand-dependent interaction of EFR 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 EFR nor BAK1. Coexpressed with the ectodomain of BAK1 fused to the C-terminal part of mCherry, elf18-induced interaction between BAK1 and EFR can drive 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 EFR 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 EFR nor BAK1. <br>
-This enables us to use this part, in coordination with the BAK1 ectodomain and mCherry, to visually capture the presence of the elf18 epitope in water samples as the elf18 pattern will induce interaciton between the receptors, causing the split-mCherry parts to rejoin and generate a funcitonal fluorescent protein.
+We were interested in testing whether coexpression of this part with BAK1 fused to the C-terminal domain of split-mCherry in <i>C. reinhardtii</i> would allow to visually capture the presence of the bacterial epitope elf18 in water samples by ligand-dependent interaction of the two receptors, an interaction which would drive the reconstitution of the split-mCherry protein.