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Revision as of 13:08, 26 October 2017 by Danakd (Talk | contribs)


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Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
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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 (Status: 500 Content-type: text/html

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) (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.


Usage and Biology

We characterized this device by transfecting HEK-293 cells with this device and two more variations of it, with pDisplay backbone including epitope of bee venom PLA antigen as a positive control, with a plasmid containing CMV promoter and GFP as a reporter gene as a negative control and also non-transfected cells. 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. The device described above is the variation that has the highest fluorescence intensity of all three variations, comparing to the positive control (Figure 1).

Sequence and Features Status: 500 Content-type: text/html

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Status: 500 Content-type: text/html

Software error:

Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 84.
Global symbol "$groups" requires explicit package name at /websites/parts.igem.org/cgi/lib/User.pm line 85.
Compilation failed in require at /websites/parts.igem.org/cgi/lib/IconBar.pm line 12.
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