Difference between revisions of "Part:BBa K2520007"
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<partinfo>BBa_K2520007 short</partinfo> | <partinfo>BBa_K2520007 short</partinfo> | ||
| − | This device is a complete system for displaying proteins on | + | 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 (<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 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. | <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 TRE promoter (<partinfo>K2520025</partinfo>) is an inducible expression promoter in mammalian cells and hGH (<partinfo>K404108</partinfo>) serves as a terminator. The addition of TRE promoter allows inducible expression | + | The TRE promoter (<partinfo>K2520025</partinfo>) is an inducible expression promoter in mammalian cells and hGH (<partinfo>K404108</partinfo>) 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 <i>Escherichia coli</i> 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, 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 | + | 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=== | ===Structure=== | ||
The structure of the TRE-Tri-Display components is shown below (figure 1). | The structure of the TRE-Tri-Display components is shown below (figure 1). | ||
[[File:Tre_tri_display.png|800px|thumb|center|Figure 1: Structure of the Tri-Display. Each display component consists of Igk leader, tag, epitope and PDGFR]] | [[File:Tre_tri_display.png|800px|thumb|center|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: <partinfo>K2520007</partinfo> - TRE-Tri Display-hGH, the plasmid being discussed, and <partinfo>K2520009</partinfo> - 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. | ||
| + | [[File:tre_tri_curve.png|500px|thumb|center|Figure 2: Weighted median of cells expressing different tags depending on different concentrations of Doxycyline]] | ||
===References=== | ===References=== | ||
Latest revision as of 13:29, 31 October 2017
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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 TRE promoter (Status: 500 Content-type: text/html
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) is an inducible expression promoter in mammalian cells and hGH (Status: 500 Content-type: text/html
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) 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).
Characterization
We co-transfected HEK-293 cells with two plasmids: Status: 500 Content-type: text/html
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- TRE-Tri Display-hGH, the plasmid being discussed, and Status: 500
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- 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.
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
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