Fluorescent Reporter Characterization
Judith Eeckman, Adam Grose, Miranda Hagen, Brittany Harwell, Swetha Pasala, Meghan
Savage, Hayley Schaefer, Loran Steinberger, Mark Stewart, Daniel Thorpe,
2011 Laura Adam, Dr. David Ball, Dr. Martha Eborall, Matthew Lux, Julie Marchand, Dr. Jean Peccoud
Laboratory of Synthetic Biology, Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA
Objectives Design Results and Analysis
1. Establish a general workflow for parts characterization We added our own attribute grammar to to design all Model
our constructs and generate their SBML files.
2. Characterize fluorescent reporters in vivo in different
contexts m d
Grammar expression degraded
Motivations Pre Pro (Tag) FP (Tag) (Tag)FP(Link) FP (Tag) Stop Suf (Tag) FP (Tag) (Tag)FP(Link) FP (Tag) Stop Vec Immature Protein Mature protein
1. Standardized workflows for Parts characterization are Single FP Fusion Single FP Fusion d
lacking in Synthetic Biology degraded
2. Context dependencies are not well understood Prefix Promoter Reporter Stop Suffix Reporter Stop Vector
3. Most fluorescent proteins have only been
E. coli
Parameter Estimation
characterized in vitro E. coli Insert Construct
Specific goals S. cerevisiae
Start Fluorescence over
S. cerevisiae Construct time of mCherry
Insert
1. Use live-cell imaging to determine maturation & E2060. The resulting
Vector curve is the R-squared
degradation rates for different fluorescent proteins Prefix Promoter Reporter Stop Suffix Reporter Stop value optimized.
2. Compare rates in eukaryotes and prokaryotes with or
without degradation tags Single FP Fusion Single FP Fusion m = 0.035 min-1
Vec
d = 0.003 min-1
3. Design and test full workflow Pre Pro (Tag) FP (Tag) (Tag)FP(Link) FP (Tag) Stop Suf (Tag) FP (Tag) (Tag)FP(Link) FP (Tag) Stop
4. Expand the use of yeast in iGEM
Workflow Characterization
Methods of Data Processing
Fluorescence in vivo
Design Fabrication Characterization Analysis Theoretical level of
fluorescence of a reporter
protein in vivo. “Averaging” “Cell-by-Cell”
Method Method
Stabilization Induction Inhibition Time
phase: Cells phase: Cells Phase: Reporter
acclimate to proliferate matures and
conditions. reporter. degrades.
Fabrication
Time Time
Fluorescent Degradation Plasmids/
Tags Organisms
Proteins Promoters Technique Calculated m and d Rates
sfGFP
YPet
No Tag LVA • Automated microfluidics Gene Organism m (min-1)
Maturation
d (min-1) Half Time*
half time
tagRFP
pLac • – custom control software
0.0082 0.0032
mOrange GFP
Sul20C • Tracks hundreds of individual cells mCitrine E. coli
±10-5
85 min
±10-5
216 min
ECFP
• Optimized time resolution 0.0014 0.0033
GFPmut3b E. coli 215 min 91 min
mCherry ±10-4 ±10-5
Venus
DsRed E. coli 0.0167 0.0047
mCherry E. coli 18 min 67 min
tdTomato ±10-5 ±10-5
mCitrine Acknowledgments *degradation could be due to photobleaching
EYFP PEST
AcGFP
CFP
All lab work was conducted at the Virginia Bioinformatics Institute
mRuby No Tag Ubi-Lys
pGal at Virginia Tech. The Team would like to thank Dr. Jean Peccoud, Achievements
tGFP mTFP1 Laura Adam, Dr. David Ball, Dr. Eborall, Matthew Lux and Julie
S. cerevisiae Marchand for their guidance in the execution of the project. The # Submitted to
project was supported through funding from the National Science # Designed # Constructed # Sequenced # Imaged # Analyzed Registry
Foundation. 28 17 13 11 3 9