FLUORESCENT REPORTER CHARACTERIZATION Judith Eeckman, Adam Grose, Miranda Hagen, Brittany Harwell, Swetha Pasala, Meghan Savage, Hayley Schaefer, Loran Steinberger, Mark Stewart, Daniel Thorpe Laura Adam, Dr. David Ball, Dr. Martha Eborall, Julie Marchand, Dr. Jean Peccoud 2011 Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Sunday, October 9, 2011
2011 Introduction TetR λ cl LacI Elowitz and Leibler 2000 Sunday, October 9, 2011
2011 Objectives 1. Characterize fluorescent reporters 2. Look for context-dependence 3. Explore the problems of parts characterization Sunday, October 9, 2011
2011 Current methods http://www.mbl.edu/news/features/shimomura.html Credit: Tom Kleindenst In vivo vs. In vitro Sunday, October 9, 2011
2011 Outline Sunday, October 9, 2011
2011 Outline Design Sunday, October 9, 2011
2011 Outline Design Sunday, October 9, 2011
2011 Outline Design Fabrication Sunday, October 9, 2011
2011 Outline Design Fabrication Sunday, October 9, 2011
2011 Outline Design Fabrication Characterization Sunday, October 9, 2011
2011 Outline Design Fabrication Characterization Sunday, October 9, 2011
2011 Outline Analysis Design Fabrication Characterization Sunday, October 9, 2011
2011 Outline Analysis Design Fabrication Characterization Sunday, October 9, 2011
2011 Outline Design Fabrication Analysis Characterization Sunday, October 9, 2011
2011 Outline Design Fabrication Analysis Characterization Sunday, October 9, 2011
Roadmap 2011 Data Name Tag Organism Design Cloning Screening Sequencing Imaging Analysis AcGFP None S. cerevisiae ECFP None S. cerevisiae GFPmut3b None S. cerevisiae mCherry(E2060) None S. cerevisiae mCherry None S. cerevisiae mCitrine None S. cerevisiae AcGFP None E. coli ECFP None E. coli GFPmut3b None E. coli mCherry(E2060) None E. coli mCitrine None E. coli AcGFP PEST S. cerevisiae ECFP PEST S. cerevisiae GFPmut3b PEST S. cerevisiae mCherry(E2060) PEST S. cerevisiae mCherry PEST S. cerevisiae mCitrine PEST S. cerevisiae AcGFP Ubi-Lys S. cerevisiae ECFP Ubi-Lys S. cerevisiae GFPmut3b Ubi-Lys S. cerevisiae mCherry(E2060) Ubi-Lys S. cerevisiae mCherry Ubi-Lys S. cerevisiae mCitrine Ubi-Lys S. cerevisiae AcGFP LVA E. coli ECFP LVA E. coli GFPmut3b LVA E. coli mCherry(E2060) LVA E. coli mCitrine LVA E. coli Fabrication Design Characterization Analysis Sunday, October 9, 2011
Parts used 2011 Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Parts used 2011 Fluorescent Proteins Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Parts used 2011 Fluorescent Proteins YPet sfGFP tagRFP mOrange GFP ECFP mCherry Venus DsRed tdTomato mCitrine EYFP AcGFP CFP mRuby mTFP1 tGFP Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Parts used 2011 Fluorescent Degradation Proteins Tags YPet sfGFP tagRFP No Tag Sul20C LVA mOrange GFP ECFP mCherry Venus DsRed tdTomato mCitrine EYFP AcGFP CFP No Tag PEST Ubi-Lys mRuby mTFP1 tGFP Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Parts used 2011 Fluorescent Degradation Plasmids/ Proteins Tags Promoters YPet sfGFP pLac tagRFP No Tag Sul20C LVA mOrange GFP ECFP mCherry Venus DsRed tdTomato mCitrine EYFP AcGFP pGal CFP No Tag PEST Ubi-Lys mRuby mTFP1 tGFP Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Parts used 2011 Fluorescent Degradation Plasmids/ Organisms Proteins Tags Promoters YPet sfGFP pLac tagRFP No Tag Sul20C LVA mOrange GFP ECFP http://www.mansfieldct.org/schools/mms/staff/hand/Bactvsvir.htm E. coli mCherry Venus DsRed tdTomato mCitrine EYFP AcGFP pGal CFP No Tag PEST Ubi-Lys mRuby mTFP1 tGFP Credit: Visuals Unlimited Inc Photographer: Thomas Deernick S. cerevisiae Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 • Parts collected - how to put them together, not just physically but digitally Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 • Parts collected - how to put them together, not just physically but digitally Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 • Parts collected - how to put them together, not just physically but digitally Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 • Parts collected - how to put them together, not just physically but digitally • Grammar helps us put parts together, and attributes associate meaning with parts, which we’ll use for modeling Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
GenoCAD 2011 • Parts collected - how to put them together, not just physically but digitally • Grammar helps us put parts together, and attributes associate meaning with parts, which we’ll use for modeling • Created custom grammar for this project - we’ll focus on the grammar now and attributes later Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
p Stop Stop Stop Our Grammar - It’s Big 2011 FP (Link) FP FP (Link) FP (Tag) FP (Tag) (Tag) (Tag) Suf (Tag) (Tag) Stop Vec Stop (Tag) FP (Tag) Pre Pro Fusion Single FP Fusion Single FP Stop Vector Reporter Suffix Reporter Stop Prefix Promoter E. coli Construct E. coli Insert Start S. cerevisiae Insert S. cerevisiae Construct Reporter Suffix Stop Reporter Prefix Promoter Stop Vector Single FP Fusion Single FP Fusion Pro (Tag) Stop Pre (Tag) (Tag) FP (Link) FP (Tag) Suf FP (Link) FP Stop Vec FP (Tag) (Tag) (Tag) FP (Tag) Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
p Stop Stop Stop Our Grammar - It’s Big 2011 FP (Link) FP FP (Link) FP (Tag) FP (Tag) (Tag) (Tag) Suf (Tag) (Tag) Stop Vec Stop (Tag) FP (Tag) Pre Pro Fusion Single FP Fusion Single FP Stop Vector Reporter Suffix Reporter Stop Prefix Promoter E. coli Construct E. coli Insert Start S. cerevisiae Insert S. cerevisiae Construct Reporter Suffix Stop Reporter Prefix Promoter Stop Vector Single FP Fusion Single FP Fusion Pro (Tag) Stop Pre (Tag) (Tag) FP (Link) FP (Tag) Suf FP (Link) FP Stop Vec FP (Tag) (Tag) (Tag) FP (Tag) Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Our Grammar - Example 2011 Goal: Design a fluorescent reporter with one fluorescent protein, one degradation tag, in E. coli- compatible vector E. coli Construct Vector Reporter Single FP Fusion Vector FP FP (Tag) (Tag) (Tag) (Link) (Tag) FP Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Our Grammar - Example 2011 Goal: Design a fluorescent reporter with one fluorescent protein, one degradation tag, in E. coli- compatible vector E. coli Construct Vector Reporter Single FP Fusion FP FP (Tag) (Tag) (Tag) (Link) (Tag) Specific FP Vector Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Our Grammar - Example 2011 Goal: Design a fluorescent reporter with one fluorescent protein, one degradation tag, in E. coli- compatible vector E. coli Construct Vector Reporter Single FP Fusion FP FP (Tag) (Tag) (Tag) (Link) (Tag) Specific FP Vector Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
Our Grammar - Example 2011 Goal: Design a fluorescent reporter with one fluorescent protein, one degradation tag, in E. coli- compatible vector E. coli Construct Vector Reporter Single FP Fusion FP FP (Tag) (Tag) (Link) (Tag) Specific Specific Tag Vector Specific FP Fabrication Analysis Design Characterization Characterization Sunday, October 9, 2011
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