Part:BBa_K2520003

TRE-Mono display:pro insulin-hGH

This device is a complete system for displaying proteins on cells' membrane. The Igk leader (BBa_K2520024) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (BBa_K2520026) is a trans-membrane domain that anchors all the components located between the IgK leader and the PDGFR itself to the membrane. HA (BBa_K2520030) and Myc (BBa_K2520031) are tags that bind to specific antibodies and provide an indirect method for verifying the display of proteins on the membrane. The protein that we chose to express on the membrane is an epitope that is known target of diabetes (BBa_K2520027) and is component related to our specific project.

The TRE promoter (BBa_K2520025) is an inducible expression promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator. The addition of TRE promoter allows inducible expression in specific conditions. We used the tet-off system. In the Tet-Off system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from Escherichia coli fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).

The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the target gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive.

Diabetes type 1

Diabetes type 1 is a chronic disease of the pancreas that is caused due to T cells attacking the insulin producing beta cells, within the pancreas. Globally, nearly 40 million people suffer from type I diabetes. This disease leads pancreatic dysfunction and insulin dependence.

Proinsulin: Proinsulin is the prohormone precursor to insulin made in the beta cells of the islets of Langerhans. Proinsulin formed by three distinct chains: The A chain, B chain, and the area connecting the two chains - C peptide.

References

(1) Wong, F. Susan, et al. "Identification of an MHC class I-restricted autoantigen in type 1 diabetes by screening an organ-specific cDNA library." Nature medicine 5.9 (1999): 1026-1031.‏

(2) Narendran, Parth, Stuart I. Mannering, and Leonard C. Harrison. "Proinsulin—a pathogenic autoantigen in type 1 diabetes." Autoimmunity reviews 2.4 (2003): 204-210.‏ APA

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 378
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1825