Team:BostonU/Future

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

Future Work

Complete priority encoder We are in the process of gathering flow cytometry data for our basic parts library to complete Phase I of the Chimera workflow. We will prioritize the testing of the tandem promoters pBad-pBetI and pLmrA-pSrpR, fusion proteins BetI-GFP and LmrA-GFP, and the origins of replication p15A and pSC101. We have begun carrying out multiplexing reactions as part of Phase II. We plan to gather data for the range of functionality of our transcriptional unit variants and begin assembling our priority encoder from its constitutive transcriptional units. Functionality of the priority encoder via flow cytometry verification will mark the completion of Phase III and the Chimera workflow.

A possible abstract genetic regulatory network design for the priority encoder. This file was generated with Eugene and shows a possible configuration of parts to acheive function. After testing each individual transcriptional unit in Phase II, Phase III will involve testing each repression arc and assembly of the final encoder.

Refine Chimera The testing of our workflow by building the priority encoder is intended to demonstrate the ability of Chimera's methodology and software tools to guide a predictive assembly of a complex genetic regulatory network. We will consider our documented difficulties in cloning, testing, and interpreting data to refine our workflow and, most importantly, define concrete metrics that must be met at each phase of the workflow. These metrics must be adaptable to any user's target device, and must therefore allow for flexibility in their definition. For example, our version of the workflow currently specifies that we screen 10 colonies per plate if we have multiplexed 5 different basic parts to allow testing of a higher range of transcriptional unit variety without excessive screening. This metric would be different for protocols that measure TU variety without multiplexing in quantity, but would still be present to ensure that an appropriate variety of gene expression is being observed. Push Chimera to the limit The software tools we employ have the capability of allowing for construction of regulatory networks with greater complexity that our priority encoder. We aim to use Chimera to construct a novel device with nonlinear behavior, which will involve controlled protein degradation and a more complex testing scheme.

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