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>Motivation and Background</h2>
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<h3>The Chimera Characterization Workflow</h3></td></tr>
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We will complete a library of basic parts currently composed of existing ribosomal binding sites and terminators from the <a href="http://cidarlab.org/" target="_blank">CIDAR Lab</a> by adding a series of <a href="https://2014.igem.org/Team:BostonU/TandemPromoters">tandem promoters</a>, <a href="https://2014.igem.org/Team:BostonU/FusionProteins">fusion proteins</a>, and <a href="https://2014.igem.org/Team:BostonU/Backbones">vector backbones</a>. These parts will 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. Data will be gathered for these TUs using 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 characterize all the parts in our library. The TASBE Tools allow for calibrated measurement of gene expression in absolute units of fluorescence, and will allow for effectively designing multi-TU genetic circuits. We hypothesize that guiding the design and construction of complex circuits with our characterization data and workflow will streamline the traditional design-build-test cycle and aid in a more efficient process for the assembly of novel devices.
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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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As a measurement team, we will also 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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<h2>Tandem Promoters</h2>
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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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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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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>Fusion Proteins</h2>
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Fusion proteins are analogous to tandem promoters. For our purposes, we fused multiple repressors with reporter proteins in order to check whether a transcriptional unit containing fusion proteins worked just as well as two transcriptional units (one with the repressor and the other with the reporter protein). These constructs were fused by adding unique fusion sites to their ends and then, ligating them using the Modular Cloning method. The objective of making this library was to help us create standard transcriptional units that could universally be used to make intricate genetic circuits.
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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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