Part:BBa_K2520007

TRE-Tri display-hGH

This device is a complete system for displaying proteins on the membrane of mammalian cells. 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 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 under 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 downstream 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 (a tetracycline analogue), 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 of the downstream gene is silenced.

Structure

The structure of the TRE-Tri-Display components is shown below (figure 1).

Figure 1: Structure of the Tri-Display. Each display component consists of Igk leader, tag, epitope and PDGFR

Characterization

We co-transfected HEK-293 cells with two plasmids: BBa_K2520007 - TRE-Tri Display-hGH, the plasmid being discussed, and BBa_K2520009 - CMV-tTA-hGH. The transfected cells expressed different levels of three tags depending on Doxycycline concentration, due to the binding of tTA to the TRE promoter.

Figure 2: Weighted median of cells expressing different tags depending on different concentrations of Doxycyline

References

Agha-Mohammadi, Siamak, et al. "Second‐generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness." The journal of gene medicine 6.7 (2004): 817-828.

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 1963