Part:BBa_K2520010

EF1a-Tri display-hGH

This device is a complete system for displaying proteins on cells' membrane. We combined three independent systems into one, which allows for the expression of three unique proteins. The Igk leader (BBa_K2520024, BBa_K2520028, BBa_K2520029) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (BBa_K2520026, BBa_K2520036, BBa_K2520037) 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), Myc (BBa_K2520031) and His (BBa_K2520032) are tags that bind to specific antibodies and provide an indirect method for verifying the display of the proteins on the membrane. The proteins that we chose to express on the membrane are three epitopes that are known targets of multiple sclerosis (BBa_K2520038, BBa_K2520039, BBa_K2520040) and are components related to our specific project. P2A (BBa_K2520033) and T2A (BBa_K1537017) are internal peptide cleavage sites that allow for the equimolar expression of multiple proteins under the same promoter.

The EF1a promoter (BBa_K2520023) is a constitutive promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator. EF1a promoter is very useful in cells where other promoters, such as CMV, are underactive or silenced (such as stem cells).

Figure 1: Fold change of Fluorescence intensity of three different epitopes that were expressed on the membrane under two promoters- EF1a (BBa_K2520023) and CMV promoter, using Flow cytometry

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 643
  Illegal XhoI site found at 1042
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 777
  Illegal AgeI site found at 151
  Illegal AgeI site found at 1315
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2900