Difference between revisions of "Team:NAU-CHINA/Overview"

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<li>Overview</li>
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                        <li>We initially targeted our research in foundational advance and therapeutics, and we hope to use this 0-1 switch for the treatment of cytokine storms produced by immunotherapy. We interviewed several Chinese Academy of Sciences researchers with our original idea. They proposed that the current treatment of cytokine storms often takes simpler and easier injection therapy, and our pathway design is relatively complex, preferring a basic component. design. So we changed our track into foundational advance.
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Our design consists of five components.<br>
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In our successfully engineered T cells, the intracellular constitutive promoter continues to express TetR and the SynNotch-TEV fusion protein secreted to the membrane. The expressed TetR binds to TetO and inhibits the expression of Bxb1and Bxb1-RDF inhibitor downstream. When the corresponding signal appears outside the cell, the extracellular domain of SynNotch-TEV senses changes, the connection with TEV is cut, TEV enters the cell, destroys the structure of TetR, and opens the expression of downstream Bxb1and Bxb1-RDF inhibitor, recombination. The enzyme Bxb1 recognizes the flipping site attB and attP, reverses the downstream Bxb1-RDF and RFP, and transforms it into attL and attR sites to initiate transcriptional expression. At this time, RFP expression, red fluorescence is detected in the cell; while the fragment is not flipped back immediately by Bxb1-RDF due to inhibition by the Bxb1-RDF inhibitor.<br>
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When the signal is weakened and the TEV protein concentration is below the threshold, TetR re-blocks the expression of Bxb1 and Bxb1-RDF inhibitor due to accumulation of expression. The inhibition of recombinase-RDF was lifted, the sequence was inverted to its original state and the expression of RFP was turned off, and the fluorescence was quenched in the cells. It can be seen that in our overall design, we have realized a resettable and more accurate 0/1 switch through the two pairs of components TetO/TetR and Bxb1/Bxb1-RDF.<br>
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After digital-analog verification, it was found that replacing the promoter downstream of TetO with the recombinase can simulate different curves. These curves have different thresholds and sudden changes, but they can function as switches that respond quickly. The feedback was fed back to the experimental team, hoping to switch to different promoters and recombinases to make more diverse switches.<br>
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After an in-depth exchange between the experimental team and the digital model team, the literature was re-selected from the promoters downstream of TetO, and UbC, EF1α, miniCMV were selected and matched with different recombinases, including TP901, Bxb1 and PhiC31, by adjusting the parameters of each part and the strength of the promoter, our whole system has formed nine choices, which can make more threshold choices and broaden the scope of application to meet the needs of different scenarios.<br>
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Revision as of 05:21, 3 October 2018

Template:2018_NAU-CHINA

header
  1. Overview
    • We initially targeted our research in foundational advance and therapeutics, and we hope to use this 0-1 switch for the treatment of cytokine storms produced by immunotherapy. We interviewed several Chinese Academy of Sciences researchers with our original idea. They proposed that the current treatment of cytokine storms often takes simpler and easier injection therapy, and our pathway design is relatively complex, preferring a basic component. design. So we changed our track into foundational advance.
      Our design consists of five components.
      In our successfully engineered T cells, the intracellular constitutive promoter continues to express TetR and the SynNotch-TEV fusion protein secreted to the membrane. The expressed TetR binds to TetO and inhibits the expression of Bxb1and Bxb1-RDF inhibitor downstream. When the corresponding signal appears outside the cell, the extracellular domain of SynNotch-TEV senses changes, the connection with TEV is cut, TEV enters the cell, destroys the structure of TetR, and opens the expression of downstream Bxb1and Bxb1-RDF inhibitor, recombination. The enzyme Bxb1 recognizes the flipping site attB and attP, reverses the downstream Bxb1-RDF and RFP, and transforms it into attL and attR sites to initiate transcriptional expression. At this time, RFP expression, red fluorescence is detected in the cell; while the fragment is not flipped back immediately by Bxb1-RDF due to inhibition by the Bxb1-RDF inhibitor.
      When the signal is weakened and the TEV protein concentration is below the threshold, TetR re-blocks the expression of Bxb1 and Bxb1-RDF inhibitor due to accumulation of expression. The inhibition of recombinase-RDF was lifted, the sequence was inverted to its original state and the expression of RFP was turned off, and the fluorescence was quenched in the cells. It can be seen that in our overall design, we have realized a resettable and more accurate 0/1 switch through the two pairs of components TetO/TetR and Bxb1/Bxb1-RDF.
      After digital-analog verification, it was found that replacing the promoter downstream of TetO with the recombinase can simulate different curves. These curves have different thresholds and sudden changes, but they can function as switches that respond quickly. The feedback was fed back to the experimental team, hoping to switch to different promoters and recombinases to make more diverse switches.
      After an in-depth exchange between the experimental team and the digital model team, the literature was re-selected from the promoters downstream of TetO, and UbC, EF1α, miniCMV were selected and matched with different recombinases, including TP901, Bxb1 and PhiC31, by adjusting the parameters of each part and the strength of the promoter, our whole system has formed nine choices, which can make more threshold choices and broaden the scope of application to meet the needs of different scenarios.