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

Using Chimera to build the encoder We employ the Chimera workflow to fill in the generic part positions in Oberortner's encoder design and assign specific parts to each of the positions. In order to begin the 3 phases of the Chimera workflow, the priority encoder must first be decomposed and the parts necessary must be identified. To begin, the encoder necessitates the use of tandem promoters and various orthogonal 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 within the priority encoder. Below, we show where we would insert fusion GFP to help us measure the device as we build this complex device. Our multiplexing results from Phase II will provide the necessary information in terms of transfer curves to determine which parts must be placed in each position to ensure the priority encoder works as intended. As a preliminary approach we have chosen to place the priority encoder on two plasmids to increase the chances of transformed cells adopting the entire device. This approach has necessitated the creation of lower copy backbones to ensure proper function.

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