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