Team:BostonU/Chimera

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         <th scope="col">As synthetic biology continues to expand, researchers are producing a greater variety of novel and innovative genetic circuits. This research revolves around a standard design-build-test cycle that defines the timeline of a project from its conception. The design and assembly of constructs depends on a thorough understanding of their individual components, making thorough part characterization data essential. The fact that there is currently little standardization in DBT workflows and poorly documented standard parts libraries represents an increasingly significant stymying factor to the growth of the field, especially as more laboratories continue to share resources and data. We seek to strengthen the traditional design-build-test cycle fundamental to synthetic biology with a formalized workflow defined by bio-design automation software tools and built upon a thoroughly characterized library of parts.</th>
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         <td scope="col">Synthetic biology research revolves around design-build-test cycles for the production of genetic devices. An effective process often depends on protocol robustness and a thorough understanding of individual genetic components. Currently, limited software integration and part characterization represent significant stymying factors to the growth of the field, particularly as researchers endeavor to construct increasingly complex devices with behavior that is difficult to predict.<br><br>
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<th scope="col"><img src="https://static.igem.org/mediawiki/2014/d/d9/Chimera_plasmid_BU14.png" height="300" width="300" alt="ChimeraPlasmid" style="float:right" style= "margin-left:10px"><br><br><capt></capt></th>
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We seek to strengthen the traditional design-build-test cycle by developing a workflow that utilizes bio-design automation software tools and builds upon a thoroughly characterized library of parts. Our group was motivated to start this project through our participation in the <a href="https://2014.igem.org/Team:BostonU/HumanPractices">6th International Workshop on Bio-Design Automation</a>, at which important questions about software integration into wet lab experiments were discussed. Our goal is to bridge the gap between software tools and the wet lab to design a more efficient synthetic biology experimental process.</td>
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<td scope="col"><img src="https://static.igem.org/mediawiki/2014/d/d9/Chimera_plasmid_BU14.png" height="300" width="300" alt="ChimeraPlasmid" style="float:right" style= "margin-left:10px"><br><br><capt></capt></td>
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<h2>General Plan</h2>
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<h3>The Chimera Characterization Workflow</h3></td></tr>
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We aim to design genetic devices made up of several transcriptional units with the help of <a href="http://eugenecad.org/" target="_blank">Eugene</a>, a design software tool developed by the <a href="http://cidarlab.org/" target="_blank">CIDAR Lab</a> at BU. Eugene is based on <a href="http://www.sbolstandard.org/" target="_blank">SBOL</a> (Synthetic Biology Open Language), an open-source standard for in silico representation of genetic designs. Eugene is a rule-based tool that allows us to specify necessary constraints for a genetic device (such as number of parts, directionality, and order), and outputs SBOL figures that indicate possible circuit topologies. From a very large design space of potential topologies for circuits, the rules specified to Eugene allow it to suggest an experimentally viable amount of designs with topologies that we may not have considered.
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The Chimera characterization workflow is intended to facilitate the predictive design of complex genetic regulatory networks. It employs a design-build-test engineering approach made unique by the inclusion of the following computational tools: <a href="http://eugenecad.org/">Eugene</a> and <a href="http://pigeoncad.org/">Pigeon</a> for designing, <a href="http://ravencad.org/">Raven</a> for assembling, and the <a href="https://synbiotools.bbn.com/">TASBE Tools</a> for testing genetic constructs. Depending on the researcher's knowledge of device design and assembly, the Chimera workflow can be adjusted in its reliance on the computational tools employed.</td></tr>
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We then plan on decomposing our large circuit topologies into individual transcriptional units (TUs) beginning with a promoter and ending with a terminator, with the goal of measuring individual component behavior with flow cytometry. Because this decomposition will result in some TUs whose expression cannot be directly measured, elements will be added to enable observability. This requires a library of <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins</a> for fluorescence as a representative of gene expression, and <a href="https://2014.igem.org/Team:BostonU/Promoters">tandem promoters</a> for induction or repression arcs. These components will allow us to compile detailed characterization data for single and double promoter transcription rate, in addition to gene expression levels.
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Our characterized promoters and genes, in addition to characterized ribosomal binding sites and terminators from the CIDAR library, will make up a basic part library for our constructs. We will also build, characterize, and add <a href="https://2014.igem.org/Team:BostonU/Backbones">new vector backbones</a> with different origins of replication for variable plasmid copy counts to our library. These new backbones will allow for protein expression better suited to the desired constructs.
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The parts in our library (promoters, RBSs, genes, terminators, and backbones) will then be cloned using the <a href="https://2014.igem.org/Team:BostonU/MoClo">MoClo</a> assembly method in multiplexing reactions to create a library of transcriptional units. Characterization data will be gathered for these TUs, which will form the basis for the assembly of our target constructs.
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We will use the data from our thoroughly characterized basic parts and TU libraries to define the combinations of TUs that are most likely to yield a desired behavior in our multi-TU constructs. We hypothesize that having a wealth of parts and data available will allow for a much more efficient workflow and a greater likelihood that the parts chosen for our constructs will allow for optimal behavior. The assembly of our circuits will be aided by <a href="http://cidarlab.org/raven/" target="_blank">Raven</a>, a web-based tool from the CIDAR lab that generates an assembly plan for genetic devices. Raven's assembly plans are designed to minimize the labor and reagent costs involved in constructing genetic devices, alleviating much of the effort in manually designing assembly plans.
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We will use flow cytometry in conjunction with the <a href="https://synbiotools.bbn.com/" target="_blank">TASBE Tools</a> developed at <a href="http://www.bbn.com/" target="_blank">BBN Technologies</a> to test the functionality of our library parts and new constructs. The TASBE Tools allow for calibrated measurement of gene expression in absolute units of fluorescence.
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<h2>Timeline</h2>
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We will first focus on the assembly and characterization of new vector backbones, tandem promoters, and fusion proteins to compose our basic parts library. We will then carry out our multiplexing reactions to build and characterize our TU library. Following the creation of our libraries, we will use Eugene to design, Raven to build, and the TASBE tools to characterize our larger, more complex circuits.
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As a measurement team, we will use our flow cytometer and the TASBE Tools to enhance the documentation of existing Registry parts. We will contribute our entire basic parts and TU libraries to the Registry to enable other synthetic biology groups to rely on well-characterized parts and methods for their research.
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<center><capt>This is a graphic depiction of where the software tools Eugene, Pigeon, Raven, and TASBE Tools enter into the classic design-build-test engineering cycle. We also have a Share branch to reflect sharing our data with the iGEM community on the Registry Pages and also through the Synthetic Biology Open Language (SBOL) sharing capabilities.</capt></center></td>
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The end goal of the Chimera workflow is to collect high quality quantitative characterization data from genetic devices that can then be used to inform the users on which parts are reliable for use in more complex devices. Currently, this workflow uses flow cytometry as a means of measuring functionality of genetic devices due to the use of the TASBE tools, which allows users to easily process flow cytometry data. <br><br>When it comes to the design and build aspects of this workflow, Chimera is unbiased when it comes to which assembly method the user selects thanks to Eugene and Raven, which are tools agnostic to assembly method.</td>
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A desired genetic device behavior and an idea of the parts required are all a researcher needs to begin using Chimera. Once these characteristics have been targeted, the workflow can be used to guide a researcher to building their intended device more efficiently. The following is a general outline of the Chimera workflow. An example of BU 2014's test case can be found on our <a href="https://2014.igem.org/Team:BostonU/Workflow">workflow</a> page, in which we test the functionality of Chimera by using it to assemble a priority encoder.<br><br><br>
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<h2>Tandem Promoters</h2>
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Tandem promoters are useful for building logic gates in complex genetic circuits. To expand the types of circuits we can build, our team added several Level 0 MoClo tandem promoter parts to our library. We designed a new fusion site, K, with the sequence ATGC. This allowed us to combine two promoters (AK and KB fusion sites) to form a level 0 AB tandem promoter MoClo part. Using the pBad, pA1LacO, pTet, and R0051 promoters, we made level 0 parts with all possible combinations.
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Latest revision as of 00:15, 18 October 2014



Project Chimera
Synthetic biology research revolves around design-build-test cycles for the production of genetic devices. An effective process often depends on protocol robustness and a thorough understanding of individual genetic components. Currently, limited software integration and part characterization represent significant stymying factors to the growth of the field, particularly as researchers endeavor to construct increasingly complex devices with behavior that is difficult to predict.

We seek to strengthen the traditional design-build-test cycle by developing a workflow that utilizes bio-design automation software tools and builds upon a thoroughly characterized library of parts. Our group was motivated to start this project through our participation in the 6th International Workshop on Bio-Design Automation, at which important questions about software integration into wet lab experiments were discussed. Our goal is to bridge the gap between software tools and the wet lab to design a more efficient synthetic biology experimental process.		 ChimeraPlasmid

The Chimera Characterization Workflow

The Chimera characterization workflow is intended to facilitate the predictive design of complex genetic regulatory networks. It employs a design-build-test engineering approach made unique by the inclusion of the following computational tools: Eugene and Pigeon for designing, Raven for assembling, and the TASBE Tools for testing genetic constructs. Depending on the researcher's knowledge of device design and assembly, the Chimera workflow can be adjusted in its reliance on the computational tools employed.
This is a graphic depiction of where the software tools Eugene, Pigeon, Raven, and TASBE Tools enter into the classic design-build-test engineering cycle. We also have a Share branch to reflect sharing our data with the iGEM community on the Registry Pages and also through the Synthetic Biology Open Language (SBOL) sharing capabilities.
 The end goal of the Chimera workflow is to collect high quality quantitative characterization data from genetic devices that can then be used to inform the users on which parts are reliable for use in more complex devices. Currently, this workflow uses flow cytometry as a means of measuring functionality of genetic devices due to the use of the TASBE tools, which allows users to easily process flow cytometry data.

When it comes to the design and build aspects of this workflow, Chimera is unbiased when it comes to which assembly method the user selects thanks to Eugene and Raven, which are tools agnostic to assembly method.
A desired genetic device behavior and an idea of the parts required are all a researcher needs to begin using Chimera. Once these characteristics have been targeted, the workflow can be used to guide a researcher to building their intended device more efficiently. The following is a general outline of the Chimera workflow. An example of BU 2014's test case can be found on our workflow page, in which we test the functionality of Chimera by using it to assemble a priority encoder.







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