Team:BostonU/Repressors

From 2014.igem.org

Revision as of 21:42, 15 October 2014

• 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

Repressor Proteins and Promoters

Why Repressor Proteins? In order to build and test the priority encoder below, we will need to create new basic parts for new repressors and their corresponding promoters. The diagram below shows an Abstract Genetic Regulatory Network, or AGRN, where only the function is outlined without any specific parts assigned to the promoters, repressors, or small molecules (with the exception of the fluorescent proteins GFP, RFP, and BFP). Our current MoClo library of parts does not contain enough orthogonal repressors and promoters to flush this design out. Based on this limitation, we began a literature search to find five new repressor and promoter pairs that could work together in one system. Early on in our search we found the 2014 article by Stanton et al. [1] where they built and tested a library of 73-TetR family repressors from various prokaryotic genomes. From these parts, they showed that 20 of them had strong transfer curves when turned on (Figure 4 below). From that subset, we chose five repressors that had desirable but slightly different transfer curves from one another: LmrA, SrpR, PhlF, BetI, and BM3R1. For our definition of desirable, we wanted to see a clear on and off function across at least two decades of REU with varying peak fluorescence as measured by Stanton et al.. Figure 4 from Stanton et al.(2014) where each repressor (noted in bottom left of each graph) was under control of an IPTG-inducible promoter and the corresponding repressor promoter controlled YFP fluorescence. Design and Assembly After choosing the five repressors and their promoters, we filled in the AGRN with specific parts as a first pass to start the design process. Without any assignments of parts, there are over 5,000 variants possible. As a first pass, we decided to assign our parts naively (see below) just to get started on designing oligos to clone the parts. We chose pTetR, pBad, and pLacI as the three small molecule promoters because we already have those in our library of parts. We have obtained plasmid DNA from AddGene for LmrA, SrpR, PhlF, BetI, and BM3R1 and have designed oligos to clone these into the MoClo CDS parts with the C fusion site on the 5' side and the D fusion site on the 3' side to fit into our current library format. It should be noted that we are not generating fusion proteins with GFP until we finish testing our current fusion proteins. Testing 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 Our Sponsors Retrieved from "http://2014.igem.org/Team:BostonU/Repressors" Recent changes What links here Related changes Special pages My preferences Printable version Permanent link Privacy policy Disclaimers