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 based on SBOL (Synthetic Biology Open Language), an open-source standard for in silico representation of genetic designs. Eugene is a rule-based tool that allows the user 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 may not have been considered by the researcher. 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 TU 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 fusion proteins for fluorescence as a representative of gene expression, and tandem promoters 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 new vector backbones 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 MoClo assembly method in multiplexing reactions to create a library of transcriptional units. These TUs will each be characterized and

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