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

 
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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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        <p class="top-title">PROJECT</p>
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         <p class="sec-title">Overview</p>
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            <h1>What is MOSFET?</h1>           
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              The metal-oxide-semiconductor field-e ffect transistor (MOSFET, MOS-FET, or MOS FET) is a type of field-effect transistor (FET). It has an insulated gate, whose voltage determines the conductivity of the device. This capacity to change conductivity by varying the amount of applied voltage can be used for amplifying or switching electronic signals. In an enhancement-mode MOSFET mode, voltage applied to the gate terminal increases the conductivity of the device. In depletion- mode transistors, voltage applied at the gate reduces the conductivity.
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            <p>The MOSFET is by far the most common transistor in digital circuits. Millions of these transistors may be included in a memory chip or microprocessor.</p>
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            <h1>Why are our cells also called MOSFET?</h1>           
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            <p>Inspired by the electronic Mosfet, we designed our project, Monitoring and Operating System Founded on Engineered T- cells, which isto be very similar to its electronic analogue Mosfet in many ways. Our Mosfet can be divided into two sub-systems, a signal detection system and a signal response system.</p>
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                <h2>Signal detection</h2>
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                <p>We use synthetic Notch (synNotch)<sup>[1]</sup>> as our signal detection receptor of the engineered T cell. When the recognition domain binds to its target antigen, it will release the effector domain (ED), which is is TEV protease. </p>
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                <h2>Signal processing</h2>
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                <p>We want to convert the extracellular analog signal into an intracellular digital signal . We use Tet operator (TetO) to achieve this goal. This part contains TetO, TEV protease from the ED of synNotch and Tet repressor (TetR) obtained from the 2017 Oxford University iGEM project. We can initially implement the filtering function by using these elements, but there is a problem of leakage because of TetO and TetR.  To solve the leakage problem, we introduced a recombinase into the system. The recombinase is capable of flipping the 5' end to the 3' end of the target sequence containing recombinase-specific sites, thereby being able to turn it on and off and realizing the zero-one switch precisely.
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                <h2>Signal output</h2>
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                <p>Finally, we achieved equal outputs by using promoters<sup>[2]</sup>> which have different strength and recombinases<sup>[3]</sup>> which have different effects. Because Since the promoter is located at downstream of TetO and upstream of recombinase, the function is self-evident (click here to see more).</p>
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                <h2>System Reset</h2>
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                <p>When the external signal disappears or falls below the threshold, our mosfet has a reset function, which is to restore the initial state. This function is achieved by a protein called recombination directionality factor (RDF).</p>
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                <h2>The relationship between electronic Mosfet and our MOSFET</h2>
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                <p>From another perspective, the original Mosfet, when loaded into a circuit, can do the same thing as our MOSFET: <br/>
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1) Constantly detect a voltage signal. <br/>
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2) Process that voltage signal (change the resistance). <br/>
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3) Output the corresponding current.  <br/>
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4) Immediately restore the original state after the externally-modified voltage is restored. <br/>
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But the principles to achieve these are completely different between electronic and molecular-genetic contexts, so the extension must be combined with the actual context.
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            <h1>References</h1>           
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            <p>[1] Morsut, L, et al. "Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. " Cell 164.4(2016):780-791.</p>
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<p>[2] Rubens, Jacob R., G. Selvaggio, and T. K. Lu. "Synthetic mixed-signal computation in living cells." Nature Communications 7(2016):11658.</p>
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<p>[3] Weinberg, B. H., et al. "Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. " Nature Biotechnology 35.5(2017):453.</p>
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Latest revision as of 00:49, 18 October 2018

Template:2018_NAU-CHINA

header
Overview

PROJECT

Overview

What is MOSFET?

The metal-oxide-semiconductor field-e ffect transistor (MOSFET, MOS-FET, or MOS FET) is a type of field-effect transistor (FET). It has an insulated gate, whose voltage determines the conductivity of the device. This capacity to change conductivity by varying the amount of applied voltage can be used for amplifying or switching electronic signals. In an enhancement-mode MOSFET mode, voltage applied to the gate terminal increases the conductivity of the device. In depletion- mode transistors, voltage applied at the gate reduces the conductivity.

The MOSFET is by far the most common transistor in digital circuits. Millions of these transistors may be included in a memory chip or microprocessor.

Why are our cells also called MOSFET?

Inspired by the electronic Mosfet, we designed our project, Monitoring and Operating System Founded on Engineered T- cells, which isto be very similar to its electronic analogue Mosfet in many ways. Our Mosfet can be divided into two sub-systems, a signal detection system and a signal response system.

Signal detection

We use synthetic Notch (synNotch)[1]> as our signal detection receptor of the engineered T cell. When the recognition domain binds to its target antigen, it will release the effector domain (ED), which is is TEV protease.

Signal processing

We want to convert the extracellular analog signal into an intracellular digital signal . We use Tet operator (TetO) to achieve this goal. This part contains TetO, TEV protease from the ED of synNotch and Tet repressor (TetR) obtained from the 2017 Oxford University iGEM project. We can initially implement the filtering function by using these elements, but there is a problem of leakage because of TetO and TetR. To solve the leakage problem, we introduced a recombinase into the system. The recombinase is capable of flipping the 5' end to the 3' end of the target sequence containing recombinase-specific sites, thereby being able to turn it on and off and realizing the zero-one switch precisely.

Signal output

Finally, we achieved equal outputs by using promoters[2]> which have different strength and recombinases[3]> which have different effects. Because Since the promoter is located at downstream of TetO and upstream of recombinase, the function is self-evident (click here to see more).

System Reset

When the external signal disappears or falls below the threshold, our mosfet has a reset function, which is to restore the initial state. This function is achieved by a protein called recombination directionality factor (RDF).

The relationship between electronic Mosfet and our MOSFET

From another perspective, the original Mosfet, when loaded into a circuit, can do the same thing as our MOSFET:
1) Constantly detect a voltage signal.
2) Process that voltage signal (change the resistance).
3) Output the corresponding current.
4) Immediately restore the original state after the externally-modified voltage is restored.
But the principles to achieve these are completely different between electronic and molecular-genetic contexts, so the extension must be combined with the actual context.

References

[1] Morsut, L, et al. "Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. " Cell 164.4(2016):780-791.

[2] Rubens, Jacob R., G. Selvaggio, and T. K. Lu. "Synthetic mixed-signal computation in living cells." Nature Communications 7(2016):11658.

[3] Weinberg, B. H., et al. "Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. " Nature Biotechnology 35.5(2017):453.

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