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| <h2 style='padding-top: 40px; font-family: "Verlag-Book"; font-size: 50px;'> | | <h2 style='padding-top: 40px; font-family: "Verlag-Book"; font-size: 50px;'> |
− | Description | + | Interlab |
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| <div class="row"> | | <div class="row"> |
− | <div class="col-md-4"> | + | <!--div align="center"> |
− | <div class="title add-animation-stopped">
| + | <h3 style='padding-top: 50px; padding-bottom: 50px;'>Advisors</h3> |
− | <p class='h2WM' style='padding-left:70px; padding-top: 0px;'>
| + | </div--> |
− | Motivation
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− | </p>
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− | </div>
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− | </div> | + | |
− | <div class="col-md-7 col-md-offset-1" style='padding-top: 0px;'>
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− | <div class="description add-animation-stopped animation-1">
| + | |
− | <p class='large' style="color:#A9A9A9;">
| + | |
− | Genetic circuits exist in great abundance in nature as complex metabolic pathways which interact
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− | in various ways to perform vital cellular processes. Synthetic biologists aim to not only understand
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− | naturally occurring circuit networks, but also to modify them or to conceptualize and build entirely
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− | new circuits.
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− | </p>
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− | </div>
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− | </div>
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| <div class="description"> | | <div class="description"> |
− | <p class='large' style="padding-left:70px;"> | + | <p class='large' style="padding-left:40px; text-indent: 50px;"> |
− | The inherent versatility of synthetic genetic circuitry has lead to a vast array of diverse applications
| + | We participated in the Interlab Measurement Study using Flow Cytometry analysis on a FACS (Fluorescence-Activated Cell Sorter) |
− | in countless fields. However, the field remains fundamentally limited by the magnitude and specificity of
| + | machine following the protocol provided by iGEM. Our results are presented here: |
− | behavioral control over genetic circuits and circuit networks. These limitations can be boiled down to two
| + | |
− | essential problems: inherent constraints to behavior based on the nature of a circuit’s constituent genes,
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− | and the inefficiency of the “design-build-test” cycle which is relied upon for the construction of effective
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− | circuit models.
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| </p> | | </p> |
− | <p class='large' style="padding-left:70px;"> | + | <p style="text-align: center; padding-top: 30px;"> |
− | The fundamental constraints of integral circuit components limit the ability to design and construct | + | <img src="https://static.igem.org/mediawiki/2016/c/c7/Updated_interlab_160902_data.svg" width="600px"> |
− | genetic circuits of arbitrary and highly specific behavior. When constructing a circuit with some intended
| + | |
− | behavior, design is limited by the available input-specific regulators to gene expression and their
| + | |
− | characteristic regulatory behavior. In order to achieve more precise behavioral control, the ability to
| + | |
− | tune expression levels of regulatory elements to some desired level is vital. This limitation highlights
| + | |
− | the need for genetic devices that can modify the behavior of arbitrary genetic circuits; implementing these
| + | |
− | devices would enable precise behavioral control invariant to the constraints of the constituent genes that
| + | |
− | make up the circuit in question [1].
| + | |
| </p> | | </p> |
− | <p class='large' style="padding-left:70px; padding-bottom:50px;"> | + | <p class='large' style="padding-left:40px; text-indent: 50px; padding-top: 40px;"> |
− | The other foundational limitation of genetic circuit construction addresses the inefficiency and
| + | In addition to participating in the Interlab Study, we also wanted to determine if there would be a noticeable difference |
− | unpredictability of the design and construction process itself. The progression from synthesizing parts
| + | between measurements taken at Midlog and measurements taken on those same samples at Saturation. |
− | into a circuit on a plasmid, to transformation and testing in vivo, is a lengthy and expensive process
| + | |
− | which furthermore is largely variable in terms of actual functionality of the final product [2].This often
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− | leads to a series of trial-and-error testing cycles whose products maintain a persistent level of uncertainty
| + | |
− | with regard to precise, predictable behavior. Although it is possible to achieve functional genetic circuits
| + | |
− | in this capacity, greater problems arise regarding the tunability of the product. The success of any genetic
| + | |
− | circuit relies on the ability to precisely tune a response to a range of input concentrations; it would therefore
| + | |
− | be desirable to obtain a reliable method for tuning circuit response, ideally without the need to rewire the
| + | |
− | internal workings of the circuit. This method would allow control over output expression to be implemented in
| + | |
− | a more rapid and predictable manner [3].
| + | |
| </p> | | </p> |
− | </div>
| + | <p style="text-align: center; padding-top: 30px;"> |
− | <div class="col-md-4">
| + | <img src="https://static.igem.org/mediawiki/2016/a/af/Midlog_vs_saturated.svg" width="600px"> |
− | <div class="title add-animation-stopped"> | + | |
− | <p class='h2WM' style='padding-left:70px;'>
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− | The Project
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− | </p>
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− | </div>
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− | </div>
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− | <div class="col-md-7 col-md-offset-1" style='padding-top: 0px;'>
| + | |
− | <div class="description add-animation-stopped animation-1">
| + | |
− | <p class='large' style="color:#A9A9A9;"> | + | |
− | Our project aims to provide a modular collection of genetic parts which can specifically and predictably
| + | |
− | tune the behavior of an arbitrary genetic circuit. This collection, which we have dubbed the “Circuit
| + | |
− | Control Toolbox,” consists of a suite of parts which can be added to the end of a given genetic circuit;
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− | each part provides a specific and independently tunable response which allows direct control over the
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− | ultimate output behavior of the circuit.
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− | </p>
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− | </div>
| + | |
− | </div>
| + | |
− | <div class="description">
| + | |
− | <p class='large' style="padding-left:70px;">
| + | |
− | The overall input/output behavior of any genetic circuit can be represented by a graph known as a
| + | |
− | transfer function, which relates concentration of input molecule to output protein expression.
| + | |
− | Likewise, any modifications to the circuit affecting input/output behavior can be visualized by a
| + | |
− | transformation of the transfer function representing the circuit. The Circuit Control Toolbox consists
| + | |
− | of three distinct tools which prompt unique behavioral changes to the circuit’s output relative to its
| + | |
− | input, and therefore generate different transformations of the circuit’s original transfer function.
| + | |
| </p> | | </p> |
− | </div>
| + | <p class='large' style="padding-left:40px; text-indent: 50px; padding-top: 40px;"> |
− | <div class="description">
| + | We noticed that although there were differences between the midlog and saturation measurements, they were not very drastic |
− | <p class='large' style="padding-left:70px;"> | + | across the different devices when looking at bulk population-level mean measurements, with the exception of the negative control. |
− | The RBS library [link to page] provides a collection of ribosome binding sites of varying strength; | + | </p> |
− | replacing the RBS within a circuit alters the translational efficiency of the output. This tool | + | <p class='large' style="padding-left:40px; text-indent: 50px;"> |
− | effectively allows for scaled changes in the magnitude of a circuit’s output response, thus adjusting | + | However, because we measured the devices with flow cytometry, we were additionally able to assess the changes in population |
− | the amplitude of the transfer function. | + | heterogeneity which occur with between midlog phase and saturation phase. We found that the midlog populations tended to be more |
| + | unimodal than the saturation populations. |
| </p> | | </p> |
− | <p class='large' style="padding-left:70px;"> | + | <p style="float: left; padding-top: 30px; padding-bottom: 30px;"> |
− | The Decoy Binding Array [link to page] tool implements molecular titration to tune the circuit’s
| + | <img src="https://static.igem.org/mediawiki/2016/f/f6/Interlab_left.png" width="500px"> |
− | sensitivity to input concentrations. This modification is accompanied by a shift in the threshold | + | </p> |
− | of the circuit’s transfer function. | + | <p style="float: right; padding-top: 30px; padding-bottom: 30px;"> |
| + | <img src="https://static.igem.org/mediawiki/2016/d/de/Interlab_right.png" width="500px"> |
| </p> | | </p> |
− | <p class='large' style="padding-left:70px;"> | + | |
− | The Synthetic Enhancer Suite [link to page] exploits a synthetically modified enhancer/promoter | + | <p class='large' style="padding-left:40px; text-align: center !important; padding-top: 500px; color: #A9A9A9; font-size: .75em !important;"> |
− | system engineered to allow genetic circuits to generate multi-state responses. In other words,
| + | Fig. 3: Representative single-cell histograms of absolute fluorescence levels for the same sample at midlog phase vs. at saturation phase |
− | circuits are prompted to produce distinct levels of output based on the concentration of input molecule.
| + | |
− | This creates a staircase-like curve in the transfer function for the circuit.
| + | |
| </p> | | </p> |
− | <p class='large' style="padding-left:70px;"> | + | <p class='large' style="padding-left:40px; text-indent: 50px; padding-top: 40px;"> |
− | Each of these tools functions orthogonally to the activity of the other tools; furthermore, | + | A possible explanation for this phenomenon is that the antibiotic selection for the reporter construct may weaken over time, |
− | each tool is independently tunable to a specific degree. By implementing and adjusting multiple | + | allowing subpopulations of cells to develop which have varying metabolic emphases on the reporter construct. |
− | tools to the desired degree, a diverse range of circuit output behaviors can be achieved,
| + | |
− | generating a plethora of unique transfer function responses.
| + | |
| </p> | | </p> |
− | <p class='large' style="padding-left:70px;"> | + | <p class='large' style="padding-left:40px; text-indent: 50px;"> |
− | The implementation of this Toolbox relies on its generalizability and consistency over any
| + | All of the FACS plots from our Interlab study are available upon request. |
− | arbitrary genetic circuit. A circuit’s relative output behavior may be influenced by the
| + | |
− | coding sequence for the output which it controls. In order to ensure that behavior remains
| + | |
− | consistent across any range of coding sequences, we offer an additional Ribozyme Insulator
| + | |
− | [link to page] tool. This ribozyme part, known as RiboJ, insulates a circuit’s promoter
| + | |
− | activity from the genetic context of the coding sequence, allowing for consistency in the
| + | |
− | levels of relative expression across multiple coding sequence insertions. The addition of
| + | |
− | RiboJ as an insulator justifies the application of any number of Toolbox components to the
| + | |
− | end of an arbitrary genetic circuit, allowing for consistent and predictable behavioral
| + | |
− | modifications [4].
| + | |
− | </p>
| + | |
− | <p class='large' style="padding-left:70px; padding-bottom:50px;">
| + | |
− | Our Circuit Control Toolbox can easily be implemented in any project concerned with the | + | |
− | behavior of genetic circuitry by working through the following sequence of events:
| + | |
| </p> | | </p> |
| | | |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| + | |
− | <b>1.</b> Visualize the original behavior of the circuit in question by constructing a characteristic transfer function.
| + | </div> |
− | </p>
| + | |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| + | |
− | <b>2.</b> Determine the appropriate Toolbox parts-to-use using our mathematical model. This model has been parameterized
| + | |
− | such that the model parameters correspond to actual physical variables (e.g. number of tetO arrays, plasmid backbone).
| + | |
− | </p>
| + | |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| + | |
− | <b>3.</b> Swap out the final protein coding sequence in the original circuit with a Ribo-J insulated repressor sequence
| + | |
− | that is compatible with the Toolbox.
| + | |
− | </p>
| + | |
− | <p class='large' style="padding-left:70px; padding-bottom: 50px;">
| + | |
− | <b>4.</b> Apply the appropriate Toolbox parts to the end of your circuit, and express your original final output protein
| + | |
− | at the end of the series of Toolbox components.
| + | |
− | </p>
| + | |
− |
| + | |
− | <p class='large' style="padding-left:70px; padding-bottom: 100px;">
| + | |
− | In this manner, future iGEM teams and synthetic biologists will be able to easily obtain higher levels of precision
| + | |
− | and control over the behavior of their genetic networks.
| + | |
− | </p> | + | |
| </div> | | </div> |
− | <div align="center">
| |
− | <h3 style='padding-top: 50px; padding-bottom: 50px;'>References</h3>
| |
− | </div>
| |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| |
− | <b>1.</b> Nielsen, A. A., Segall-Shapiro, T. H., & Voigt, C. A. (2013). Advances in genetic circuit design:
| |
− | Novel biochemistries, deep part mining, and precision gene expression. Current Opinion in Chemical Biology,
| |
− | 17(6), 878-892. doi:<a>http://dx.doi.org/10.1016/j.cbpa.2013.10.003</a>
| |
− | </p>
| |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| |
− | <b>2.</b> Sun, Z. Z., Yeung, E., Hayes, C. A., Noireaux, V., & Murray, R. M.Linear DNA for rapid prototyping of
| |
− | synthetic biological circuits in an escherichia coli based TX-TL cell-free system. ACS Synthetic Biology, (6), 387.
| |
− | doi:10.1021/sb400131a
| |
− | </p>
| |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| |
− | <b>3.</b> Lucks, J. B., Qi, L., Whitaker, W. R., & Arkin, A. P. (2008). Toward scalable parts families for predictable
| |
− | design of biological circuits. Current Opinion in Microbiology, 11(6), 567-573. doi:<a>http://dx.doi.org/10.1016/j.mib.2008.10.002</a>
| |
− | </p>
| |
− | <p class='large' style="padding-left:70px; padding-bottom: 30px;">
| |
− | <b>4.</b> Lou, C., Stanton, B., Chen, Y., Munsky, B., & Voigt, C. A. (2012). Ribozyme-based insulator parts buffer
| |
− | synthetic circuits from genetic context. Nature Biotechnology, (30), 1137-1142.
| |
− | </p>
| |
| </div> | | </div> |
| </div> | | </div> |