Team:BostonU/Chimera

From 2014.igem.org

• TEAM
• PROJECT
  • Overview
  • Priority Encoder
  • MoClo Assembly Method
  • Low Copy Backbones
  • Tandem Promoters
  • Fusion Proteins
  • Repressor Proteins
  • Multiplexing
  • BioDesign Automation Tools
  • Future Work
• ACHIEVEMENTS
  • Data Collected
  • Parts Submitted
  • Chimera Workflow
  • Chimera Example
  • Medal Fulfillment
• NOTEBOOK
  • Training
  • Protocols
  • Backbones
  • Tandem Promoters and Repressors
  • Fusion Proteins
• CONSIDERATIONS
  • Collaborations
  • Measurement Track
  • Safety
  • Interlab Study
  • NEGEM
  • Policy and Practices
• ACKNOWLEDGEMENTS

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.

General Plan We aim to design genetic devices made up of several transcriptional units with the help of Eugene, a design software tool developed by the CIDAR Lab at BU. 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 basic parts library made up of characterized ribosomal binding sites and terminators from the CIDAR library, in addition to newly-built fusion proteins, tandem promoters, and new vector backbones. These components will allow us to compile detailed characterization data for single and double promoter transcription rate, in addition to gene expression levels. The parts in our basic library (promoters, RBSs, genes, terminators, and backbones) will then be cloned using the MoClo 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 Raven, a web-based tool from the CIDAR lab that generates assembly plans to minimize the labor and reagent costs involved in constructing genetic devices. We will use flow cytometry in conjunction with the TASBE Tools developed at BBN Technologies 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. Timeline 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. 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. 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.

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