Difference between revisions of "Part:BBa K3617001"

Revision as of 19:23, 24 October 2020

sgp130(D1-D3)-Cub

TThis biobrick is a part of a two-protein system in Saccharomyces cerevisiae which is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1515
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

From N- to C-terminal the protein includes; An endoplasmatic reticulum import signal peptide from the Saccharomyces cerevisiae cell wall integrity and stress response component 1 (Wsc1) receptor in S. cerevisiae, the first three domains of the soluble isoform of human interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane domain from Wsc1, the C-terminal part of the split version of ubiquitin and a synthetic transcription factor LexA-VP16. LexA is a DNA binding domain from Escherichia Coli and VP16 is a transcriptional activation domain from Herpes simplex virus Type 1. Together LexA-VP16 functions as an orthogonal transcription in S. cerevisiae. Between the extracellular sgp130 domains and the transmembrane domain, a (2x)GGGGS-linker was added. Between the transmembrane domain and the C-terminal split ubiquitin domain, two basic amino acids (KR), and the (2x)GGGGS-linker was added.

structure and function

The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing the interleukin-1, interleukin-6 and interleukin-10 receptor modules of our biosensors at the plasma membrane of S. cerevisiae as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the C-terminal half of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane of S. cerevisiae.

The biobrick is intended to work together with BBa_K3617000 which possesses the soluble IL-6 receptor extracellularly and the N-terminal half of split ubiquitin. The biobrick and BBa_K3617000

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 <partinfo>BBa_K3617001 short</partinfo>
-This biobrick is a part of a two-protein system that is designed for detection of human IL-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).
+TThis biobrick is a part of a two-protein system in <i>Saccharomyces cerevisiae</i> which is designed for detection of human interleukin-6 and transduction of the signal by means of a reconstituted ubiquitin. Development of split-ubiquitin as a tool for study of protein-protein interactions in vivo was first published in 1994 and has been an essential feature in biologists’ toolbox ever since (source: https://www.pnas.org/content/pnas/91/22/10340.full.pdf). A specific mutation in the N-terminal part protects it from binding spontaneously to the C-terminal part, however, reassociation can be facilitated by binding of a pair of proteins to which ubiquitin parts are fused. Human signal transducer gp130 was expressed in yeast for the first time in 1997 and further improvements paved the way to our own chimeric transmembrane proteins (source: https://pubmed.ncbi.nlm.nih.gov/9271090/).
 <h2><span class='h3bb'>Sequence and Features</span></h2>
 <partinfo>BBa_K3617001 SequenceAndFeatures</partinfo>
-This biobrick consists of multiple parts, from N- to C-terminal the part includes; An endoplasmatic reticulum import signal peptide from the <i>Saccharomyces cerevisiae</i> cell wall integrity and stress response component 1 (Wsc1) receptor in <i>S. cerevisiae</i>, the first, second and third domain of the soluble isoform of human interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane receptor of Wsc1, the C-terminal part of the split version of ubiquitin and a synthetic transcription factor LexA-VP16. Between the three domains of sgp130 and the transmembrane domain, a GGGGS-linker was added. Between the transmembrane domain and the C-terminal split ubiquitin domain, two basic amino acids (KR), and the GGGGS-linker was added.
+From N- to C-terminal the protein includes; An endoplasmatic reticulum import signal peptide from the <i>Saccharomyces cerevisiae</i> cell wall integrity and stress response component 1 (Wsc1) receptor in <i>S. cerevisiae</i>, the first three domains of the soluble isoform of human interleukin-6 co-receptor soluble glycoprotein 130 (sgp130), the transmembrane domain from Wsc1, the C-terminal part of the split version of ubiquitin and a synthetic transcription factor LexA-VP16. LexA is a DNA binding domain from Escherichia Coli and VP16 is a transcriptional activation domain from Herpes simplex virus Type 1. Together LexA-VP16 functions as an orthogonal transcription in <i>S. cerevisiae</i>. Between the extracellular sgp130 domains and the transmembrane domain, a (2x)GGGGS-linker was added. Between the transmembrane domain and the C-terminal split ubiquitin domain, two basic amino acids (KR), and the (2x)GGGGS-linker was added.
-A part of the protein constitutes the C-terminal half of the split ubiquitin molecule, which is fused to the reporter protein cassette LexA-VP16. Ubiquitin can be split into two parts, being the N-terminal and C-terminal ubiquitin that are able to reassociate in vivo to form active ubiquitin. This complementation was utilized so that upon interleukin-6 forming a heterotrimer with the gp130 domains of this biobrick and the IL-6 receptor domains of <bbpart>BBa_K3617000</bbpart>, the whole ubiquitin-molecule is formed, leading to activation of an intracellular deubiquitinase, thereby leading to release of Cub-bound LexA/VP16 . LexA is a DNA binding domain from Escherichia Coli (EG10533 (EcoCyc); P0A7C2 (UniProt)) and Herpes simplex virus Type 1 VP16 gene is a transcriptional activation domain. Upon complementation of split ubiquitin, the LexA-VP16 transcription factor is released and transported into the nucleus where it triggers reporter expression by promoter binding.
 <h2>structure and function</h2>
-The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing interleukin-1, interleukin-6 and interleukin-10 receptors at the plasma membrane of <i>S. cerevisiae</i> as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the N-terminal part of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane of <i>S. cerevisiae</i>.
+The signal peptide and transmembrane domain constitute the backbone of the modular framework of the UCopenhagen 2020 team (CIDosis). These are used for localizing the interleukin-1, interleukin-6 and interleukin-10 receptor modules of our biosensors at the plasma membrane of <i>S. cerevisiae</i> as type I single pass transmembrane proteins. As a type I transmembrane protein, the soluble interleukin receptor domains localizes extracellularly while the C-terminal half of the split protein is intracellular. Ivanusic et al. (citation) introduced the use of the signal peptide and transmembrane domain in a split-ubiquitin system for screening for PPIs at the plasma membrane of <i>S. cerevisiae</i>.
-The two fibronectin type III soluble interleukin-6 receptor subunit alpha domains mediates the binding of the receptor to interleukin-6, as demonstrated on figure 1. The outer Ig-like domain of the receptor mediates other functions of the receptor (Vollmer et al. (which year) PMID: 10406952). The biobrick is intended to work together with <bbpart>BBa_K3617000<bbpart> that possesses the outer three domains of sgp130 extracellularly. Intracellularly,  also consists of the synthetic transcription factor (LexA/VP16?) linked to the C-terminal of the split ubiquitin domain. BBa_K3617001 and <bbpart>BBa_K3617000<bbpart> localizes to the same compartment, being the plasma membrane of <i>S. cerevisiae</i>, but will only associate upon presence of IL-6. When IL-6 is present, the extracellular domains of the IL-6R and sgp130 will associate into a heterotrimer consisting of IL-6, IL-6R, and sgp130.
+The biobrick is intended to work together with <bbpart>BBa_K3617000</bbpart> which possesses the soluble IL-6 receptor extracellularly and the N-terminal half of split ubiquitin. The biobrick and <bbpart>BBa_K3617000<bbpart> localizes to the same compartment, the plasma membrane of <i>S. cerevisiae</i>, but will only associate upon presence of IL-6. When IL-6 is present, the extracellular domains of the IL-6R and sgp130 will associate into a heterotrimer consisting of IL-6, IL-6R, and sgp130. When the trimer is formed, the N and C terminal of ubiquitin come together intracellularly. Subsequently endogenous deubiquitinase in the cytosol of <i>S. cerevisiae</i> will cleave off the synthetic transcription factor frem the C-terminal of the reconstituted ubiquitin molecule.
+Upon complementation of split ubiquitin, the LexA-VP16 transcription factor is cleaved from the C-terminal of ubiquitin and transported into the nucleus where it triggers reporter expression by promoter binding.
 <h2>Sequence optimization</h2>