Difference between revisions of "Part:BBa K2520007"

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TRE-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_K2520034) 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 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.

Structure

The structure of the TRE-Tri-Display components is shown below.

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

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