Team:MIT/Motivation

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<h1>Motivation</h1>
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<h2>RNA-based Molecular Computation</h2>
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<p>Our project aims to combine the <b>modular parts</b> of synthetic biology with the exponential growth in <b>logic circuit complexity</b> of nucleic-acid based molecular computation to create <b>RNA circuits in mammalian cells</b>.</p>
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<p>Nucleic-acid based circuitry has several advantages over traditional transcription-based topologies:</p>
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<li><b>No need for specialized parts.</b>
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<br>We can design circuit parts from the ground up instead of searching for suitable orthogonal proteins.
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<li><b>Large combinatorial space.</b>
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<br>We can generate and iterate unlimited parts and filter for constraints, such as percentage of Cs or Gs.
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<li><b>Minimal metabolic load.</b>
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<br>Nucleic acid parts are fully functional and do not need to be translated into proteins.
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<li><b>Much smaller nucleotide footprint.</b>
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<br>RNA parts require much fewer bases than the mRNAs coding for protein parts.
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<li><b>Direct interfacing with mRNA, miRNA, etc.</b>
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<br>The use of mammalian cells provides us with access to various levels of regulation, such as the RNA-interference pathway.
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<h2>RNA Strand Displacement</h2>
 
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<p>Our system of RNA circuitry in mammalian cells utilizes the mechanism of <b>RNA strand displacement</b>, a novel nucleic-acid based molecular computation tool that can be used to <b>process genetic information</b>. </p>
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<p>Qian and Winfree (Science 2011) demonstrated the viability of DNA strand displacement as a scalable mechanism for performing complex digital logic in vitro. They constructed complex AND and OR logic gates by utilizing elementary DNA strand displacement reactions known as seesawing, thresholding, and reporting.</p>
 
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<p>The basic technique of DNA strand displacement involves three single stranded DNA molecules, as shown in the diagram below. A gate strand and output strand exist as a  complex that is partially bound through complementary Watson-Crick base-pairing within the S2 binding domain. The gate strand also contains an open, unbound domain called a toehold region, T*. An input strand with a free complementary toehold region, T, can bind to the toehold region on the gate strand, and subsequently displace the output strand to yield an input-gate complex. The output strand could hypothetically be used as an input for a downstream gate-output complex. </p>
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<h2>Application Space</h2>
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<p>One potential synthetic biology application of our system is <b>detection of cancer state in mammalian cells.</b> </p>
 
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<p>The first step, sensing the cancer state, can be achieved using an engineered mRNA sensor. The state-of-the-art circuit with this function, (1 Multi-Input RNAi-Based Logic Circuit for Identification of Specific Cancer Cells, Xie et al. Science 2011) requires at least five composite parts for sensing high and low mRNA concentrations.</p>
 
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<p>The next step involves processing information from these five separate inputs, including inversion of signals, into a single signal that tells the cell to upregulate fluorescent protein production. This requires another set of promoters for each sensed mRNA and a repression system to produce the correct logic.</p>
 
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<p>The last step is induction of expression of the signal protein, which requires yet another unique promoter/protein pairing.</p>
 
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<p>We compared traditional, promoter-based synbio logic with a novel strategy from the field of DNA computing: strand displacement.
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Parts required (see below for explanation):
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<li>5 input sensor modules
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<li>5 processor modules (one for each input)
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<li>1 actuation module
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<b style="font-size:18px">Promoter-based logic</b>
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<i>Maximum number of promoters found in published synbio circuits, over time.</i>
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<br> <i> Purnick and Weiss. The second wave of synthetic biology: from modules to systems. Nature Reviews Molecular Cell Biology 10, 410-422 (June 2009) </i>
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<b style="font-size:18px">Strand displacement-based logic</b>  
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<i>Maximum number of gates found in published strand displacement-like systems from the Winfree group, over time.</i>
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<h1>Background and Motivation</h1>
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Promoter-protein pairs required: 11 (one for each module)
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<b>Max achieved in literature: 6</b>
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Strands of nucleic acid required: 80
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<h4>In the near future, biological circuits will be much more <b>modular</b> and <b>sophisticated</b> than they are now, with a ten-fold <b>smaller nucleotide footprint</b>. </h4>
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Size of each promoter-protein pair: ~1,600 bp
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<h3>The Enabling Technology: Toehold-Mediated Strand Displacement</h3>
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Length of each strand: ~40 bp
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A <b>gate strand</b> and <b>output strand</b> exist as a complex that is <b>partially bound</b> through complementary Watson-Crick base-pairing within the S2 binding domain. The gate strand also contains an <b>open, unbound domain</b> called a <b>toehold</b> region, T*. An input signal strand with a free complementary toehold region, T, can bind to the toehold region on the gate strand, and subsequently displace the output strand to yield an input-gate complex and a free output signal strand. This is called a <b>toehold-mediated strand displacement reaction</b>. The output signal strand can be used as an input signal for a downstream gate-output complex, enabling sophisticated interactions which yield full logic circuits.
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<h3>Background</h3>
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<a href="http://www.sciencemag.org/content/332/6034/1196.abstract">Qian and Winfree (<i>Science</i> 2011)</a> utilized DNA computation to create AND and OR logic gates <i>in vitro</i>. They constructed a sophisticated binary square root circuit using these gates:
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<div style="text-align: right; margin-right: 8em;"><em>Image courtesy of Lulu Qian.</em></div>
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</br>Each of these:
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</br><img src="https://static.igem.org/mediawiki/2012/6/6c/MIT_Curly_strand_1.png" height=30> <img src="https://static.igem.org/mediawiki/2012/3/36/MIT_Curly_strand_2.png" height=15>
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</br>undergoes the same <b>toehold-mediated strand displacement reaction</b>. These reactions are fully modular and can be scaled to circuits of any degree of sophistication.
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<h3 id="s2">Motivation for Bringing Strand Displacement to Mammalian Synthetic Biology</h3>
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<li><b>More sophisticated circuits with smaller nucleotide footprint</b>
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<center><img src="https://static.igem.org/mediawiki/2012/b/bc/MIT_Transcription_versus_strand_displacement_circuits.png" width=350></center>
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</br> Sophistication of traditional transcription-translational circuits has grown linearly over the past 10 years, while sophistication of strand-displacement circuits has grown nearly exponentially.
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<li><b>Simple combinatorial design space</b>
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</br>With 4 nucleotides, we can create a nearly infinite number of orthogonal sequences leading to orthogonal parts.
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<li><b>Ease of composition</b>
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</br>The input motif matches the output motif allowing for modular cascading reactions.
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<li><b>Tunability</b>
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</br>We can set arbitrary digital signal thresholds by varying the concentration of circuit species. We can also achieve signal amplification by including a fuel molecule.
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<h4>RNA versus DNA</h4>
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<b>Total size of circuit: ~17,600 bp</b>
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<b>Total size of circuit: ~3,200 bp</b>
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An information processing system is of limited use without dynamic production. RNA is a good medium because it can be continually produced from a few initial DNA parts. As in nature, DNA acts as the information-storage medium, and RNA acts as the information-processing medium. We can transfect DNA parts into mammalian cells to co-opt existing cellular machinery to produce our RNA parts.
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</br>Our RNA parts can then interact with the cell through <a href="https://2012.igem.org/Team:MIT/Sensing">sensing</a> and <a href="https://2012.igem.org/Team:MIT/Actuation">actuation</a>. Endogenous cellular RNAs can act as inputs, and we can actuate by knocking down endogenous cellular RNAs.
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Latest revision as of 03:55, 27 October 2012

iGEM 2012

Background and Motivation

In the near future, biological circuits will be much more modular and sophisticated than they are now, with a ten-fold smaller nucleotide footprint.

The Enabling Technology: Toehold-Mediated Strand Displacement




A gate strand and output strand exist as a complex that is partially bound through complementary Watson-Crick base-pairing within the S2 binding domain. The gate strand also contains an open, unbound domain called a toehold region, T*. An input signal strand with a free complementary toehold region, T, can bind to the toehold region on the gate strand, and subsequently displace the output strand to yield an input-gate complex and a free output signal strand. This is called a toehold-mediated strand displacement reaction. The output signal strand can be used as an input signal for a downstream gate-output complex, enabling sophisticated interactions which yield full logic circuits.

Background

Qian and Winfree (Science 2011) utilized DNA computation to create AND and OR logic gates in vitro. They constructed a sophisticated binary square root circuit using these gates:
Image courtesy of Lulu Qian.


Each of these:

undergoes the same toehold-mediated strand displacement reaction. These reactions are fully modular and can be scaled to circuits of any degree of sophistication.

Motivation for Bringing Strand Displacement to Mammalian Synthetic Biology

  • More sophisticated circuits with smaller nucleotide footprint

    Sophistication of traditional transcription-translational circuits has grown linearly over the past 10 years, while sophistication of strand-displacement circuits has grown nearly exponentially.
  • Simple combinatorial design space
    With 4 nucleotides, we can create a nearly infinite number of orthogonal sequences leading to orthogonal parts.
  • Ease of composition
    The input motif matches the output motif allowing for modular cascading reactions.
  • Tunability
    We can set arbitrary digital signal thresholds by varying the concentration of circuit species. We can also achieve signal amplification by including a fuel molecule.

RNA versus DNA

An information processing system is of limited use without dynamic production. RNA is a good medium because it can be continually produced from a few initial DNA parts. As in nature, DNA acts as the information-storage medium, and RNA acts as the information-processing medium. We can transfect DNA parts into mammalian cells to co-opt existing cellular machinery to produce our RNA parts.

Our RNA parts can then interact with the cell through sensing and actuation. Endogenous cellular RNAs can act as inputs, and we can actuate by knocking down endogenous cellular RNAs.

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