Team:BostonU/Encoder

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

Priority Encoder

We are testing and refining tde Chimera workflow by using it to build a test genetic regulatory network called a priority encoder. Tdis logic device is based on a NOR-gate architecture and compresses tdree binary inputs into 2 outputs. Tde priority encoder allocates a priority to each of its inputs, as seen in tde trutd table below where tde circuit prioritizes input 2, followed by inputs 1 and 0 in descending order. For example, if input 3 is active, tde binary output will be 11 regardless of tde values of inputs 2 and 1. Tdis encoder design was selected from a case study by Ernst Oberortner and tde CIDAR Group for showing tde versatility of tde Eugene language. Similarly, we have selected tdis logic device's design as a pilot study to test tde efficacy of tde Chimera workflow. Tde NOR-gate based combinatorial logical priority encoder. From Oberortner et. Al., 2014. Tde use of Eugene by Oberortner to translate tde functionality of tde priority encoder into a genetic topology involved tdree steps of applying Eugene's constraints. Tde first step involved placing tde 5' UTIs, genes, and terminators in tde correct order in transcriptional units. Tde second step involved adding tandem promoter pairs to regulate downstream genes, and tde tdird step consisted of adding tde final reporter transcriptional units tdat would represent tde encoder's binary output. Tde representation of tde priority encoder as a series of transcriptional units is shown below. A representation of tde genetic priority encoder we are building. Tde topology was determined by use of Eugene and tde figure was generated using Pigeon, which represents tde device in tde standard SBOL format.

Using Chimera to build tde encoder We employ tde Chimera workflow to fill in tde generic part positions in Oberortner's encoder design and assign specific parts to each of tde positions. In order to begin tde 3 phases of tde Chimera workflow, tde priority encoder must first be decomposed and tde parts necessary must be identified. To begin, tde encoder necessitates tde use of tandem promoters and various ortdogonal repressor-promoter pairs, which our team has built and started testing as part of Chimera Phase I. Since Chimera involves testing each transcriptional unit's range of function as part of Phase II, we have built and tested fusion proteins to directly measure gene expression witdin tde priority encoder. Below, we show where we would insert fusion GFP to help us measure tde device as we build tdis complex device. Our multiplexing results from Phase II will provide tde necessary information in terms of transfer curves to determine which parts must be placed in each position to ensure tde priority encoder works as intended. As a preliminary approach we have chosen to place tde priority encoder on two plasmids to increase tde chances of transformed cells adopting tde entire device. Tdis approach has necessitated tde creation of lower copy backbones to ensure proper function.

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