Team:BostonU/Chimera

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Project Chimera

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.

Motivation and Background

We will complete a library of basic parts currently composed of existing ribosomal binding sites and terminators from the CIDAR Lab by adding a series of tandem promoters, fusion proteins, and vector backbones. These parts will will then be cloned using the MoClo 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 TASBE Tools developed at BBN Technologies 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.

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.

Tandem Promoters

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.

Fusion Proteins

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.

Our Sponsors

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-<h2>General Plan</h2>
+<h2>Motivation and Background</h2>
 <br>
 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.