Difference between revisions of "Part:BBa K3617001"
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<partinfo>BBa_K3617001 short</partinfo>
 
<partinfo>BBa_K3617001 short</partinfo>
  
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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/).
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This 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>
 
<h2><span class='h3bb'>Sequence and Features</span></h2>

Revision as of 19:23, 24 October 2020

sgp130(D1-D3)-Cub

This 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