Team:BostonU/Multiplexing

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

Multiplexing

Why multiplexing? As the key method of Phase II of the Chimera workflow, multiplexing is being employed by our group to determine the full range of function of our transcriptional units (TUs). We define full range of function as having a set of TUs with one varied constituent part, whose transfer curves display a difference across at least two decades of MEFLs with varying peak fluorescence as measured by flow cytometry. This wide range is desirable and essential to the robustness of the workflow because it provides the constitutive parts of a device with diverse expression levels. Having this diversity makes having a desired combination of parts more likely, which can more easily result in a desired device behavior. We have selected multiplexing as our tool to achieve this diversity because of its compatibility with our MoClo assembly method, which facilitates the quick replacement of any set of parts in a transcriptional unit.
Building multiplexed transcriptional units Our first multiplexing reactions vary the ribosomal binding sites (RBSs) of transcriptional units. We began by creating a Eugene file (found here) to plan our first reaction. By specifying constraints on the number and order of parts, in addition to the number of RBSs to be multiplexed, Eugene outputted five possible layouts from a possible design space of 65,536 possibilities. A MoClo reaction was prepared to multiplex the RBSs B0030, B0031, B0032, B0033, and B0034 in a TU with repressible promoter I13453, GFP E0040, terminator B0015, and origin of replication pMB1 in a MoClo Level 1 backbone. This set of parts was used as a starting point for directly observing the effect of changing a ribosomal binding site with a repressible promoter, which is a layout similar to what will be present in the priority encoder. A second MoClo reaction was prepared to multiplex the remaining RBSs from our lab: BCD1, BCD2, BCD8, BCD12, and BCD13 in the same TU layout.	Pigeon CAD images of the two initial multiplexing MoClo reactions that were carried out.
Because MoClo requires an equal amount of DNA for each part, two RBS master mixes were prepared with 8fmol of each individual RBS, totaling 40fmol of RBS mix. Following MoClo, blue-white screening is carried out to pick colonies that incorporated the new plasmid. We have decided to begin by picking 10 colonies per plate to increase the possibility of picking at least one colony per RBS. Testing of multiplex reactions Testing of our multiplex reactions will follow our flow cytometry workflow and our testing scheme with the appropriate single and double expression fluorescence controls for use with the TASBE Tools.
References [1] B. Stanton, A. Nielsen, A. Tamsir, K. Clancy, T. Peterson & C. Voigt (2014). "Genomic mining of prokaryotic repressors for orthogonal logic gates." Nature Chemical Biology 10: 99-105. doi:10.1038/nchembio.1411

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