Team:BostonU/Chimera

         <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>

<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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<h2>General Plan</h2>

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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.

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/TandemPromoters">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.

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.

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.

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.

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.

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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<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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<h2>Fusion Proteins</h2>

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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