Difference between revisions of "Part:BBa K2520008"

Revision as of 15:08, 27 October 2017

CMV-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 CMV promoter (BBa_K1119006) is a constitutive expression promoter in mammalian cells and hGH (BBa_K404108) serves as a terminator.

Structure

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

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

Characterization

We characterized this device by transfecting HEK-293 cells with this device, in comparison to non-transfected cells (NT). We incubate the transfected cells with specific antibodies that bind to three different tags (HA, Myc and His) conjugated to chromophores and measured the fluorescence intensity emitted. (Figure 2).

Figure 2: Weighted median of optimized Tri-Display components

Sequence and Features