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