Difference between revisions of "Part:BBa K2520008"
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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 (<partinfo>K2520024</partinfo>, | 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 (<partinfo>K2520024</partinfo>, | ||
| − | <partinfo>K2520028</partinfo>, <partinfo>K2520029</partinfo>) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (<partinfo>K2520026</partinfo>, <partinfo>K2520036</partinfo>, <partinfo>K2520037</partinfo>) is a trans-membrane domain that anchors all the components located between the | + | <partinfo>K2520028</partinfo>, <partinfo>K2520029</partinfo>) is a short signal peptide that prompts the translocation of a protein to the cellular membrane. PDGFR (<partinfo>K2520026</partinfo>, <partinfo>K2520036</partinfo>, <partinfo>K2520037</partinfo>) is a trans-membrane domain that anchors all the components located between the Igk leader and the PDGFR itself to the membrane. HA (<partinfo>K2520030</partinfo>), Myc (<partinfo>K2520031</partinfo>) and His (<partinfo>K2520032</partinfo>) 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 (<partinfo>K2520038</partinfo>, <partinfo>K2520039</partinfo>, <partinfo>K2520040</partinfo>) and are components related to our specific project. P2A (<partinfo>K2520033</partinfo>) and T2A (<partinfo>K1537017</partinfo>) are internal peptide cleavage sites that allow for the equimolar expression of multiple proteins under the same promoter. |
The CMV promoter (<partinfo>K1119006</partinfo>) is a constitutive expression promoter in mammalian cells and hGH (<partinfo>K404108</partinfo>) serves as a terminator. | The CMV promoter (<partinfo>K1119006</partinfo>) is a constitutive expression promoter in mammalian cells and hGH (<partinfo>K404108</partinfo>) serves as a terminator. | ||
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===Characterization=== | ===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 | + | 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). |
[[File:Cmv-tri-display-graph.png|500px|thumb|center|Figure 2: Weighted median of optimized Tri-Display components]] | [[File:Cmv-tri-display-graph.png|500px|thumb|center|Figure 2: Weighted median of optimized Tri-Display components]] | ||
Latest revision as of 11:43, 31 October 2017
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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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) is a trans-membrane domain that anchors all the components located between the Igk leader and the PDGFR itself to the membrane. HA (Status: 500 Content-type: text/html
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), Myc (Status: 500 Content-type: text/html
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) and His (Status: 500 Content-type: text/html
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) 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 (Status: 500 Content-type: text/html
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) and are components related to our specific project. P2A (Status: 500 Content-type: text/html
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) and T2A (Status: 500 Content-type: text/html
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) are internal peptide cleavage sites that allow for the equimolar expression of multiple proteins under the same promoter.
The CMV promoter (Status: 500 Content-type: text/html
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) is a constitutive expression promoter in mammalian cells and hGH (Status: 500 Content-type: text/html
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) serves as a terminator.
Structure
The structure of the Tri-Display components is shown below.
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).
Sequence and Features
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