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1. Tri-Stable Switch

2. Lead Detector

3. iGEM Community

Tri-Stable SwitchAdam Emrich, Norris Hung, Kyle Schutter

Inspiration

Architecture

Modeling

Applying the Model

Status

Inspiration

Gardner, Cantor, & Collins in Nature 2000

Our Role Model

n-Stable Switch

“If I have seen a little further it is by standing on the shoulders of Giants." - Isaac Newton

- Apply their logic

- Apply their model

- Use iGem Parts

Model Product

Architecture

Black Box

aTc

IPTG

ArabinoseGFP

RFP

YFP

PAraC/BADPAraC/BAD TetRTetRLacI

LacI

LacI

LacI

TetR

TetRAraCAraC

AraCAraC

PLac

I

PLac

I

PTetRPTetR

GFPGFP

YFPYFP

RFPRFP

L-arabinose

IPTG

aTc

Modeling

β = cooperativity of repressionα = repressor production ratex = repressor1y = repressor2z = repressor3

€

dy

dt=

α 11+ x β1

+α 3

1+ zβ 3− y

€

dz

dt=

α 11+ x β1

+α 2

1+ y β 2− z

€

dx

dt=

α 21+ y β 2

+α 3

1+ zβ 3− x

The Model

Change in

Repressor

Change in

Repressor

Less Repressio

n by Other

Repressors

Less Repressio

n by Other

Repressors

Degradation

Degradation

Repressor

Production

Applying the model

Repressor Concentration

dX/dt

β=1

β=10

β=2β=3

Rate of Change of Repressor vs Repressor Concentration

for Different Cooperativity Levelsd

Y

(X or Z)

Experimentally Determine Model Parameters

How Can the Tri-Stable Switch be used?

C

A

B

GFPGFP

YFPYFP

RFPRFP

Protein 1

Protein 2

Protein 3

Applications

Basic Circuit Component

Memory (Trinary?)

Drug Delivery

Cell Differentiation

α=?

Summer GoalsDesign Genetic Architecture

Derive Model

Experimentally Determine Model Parameters

Test and Build Switch

€

dx

dt=

α 21+ y β 2

− x

Summer Accomplishments

tests designed

ligations underway

Lead DetectorDeepa Galaiya, Jeff Hofmann, Rohan Maddamsetti

Inspiration

Architecture

Part Characterization

Progress

Inspiration

Contaminated Water: __%

Clean Water: __%

Contaminated Water: 40%

Clean Water: 60%

WHO

Inspiration

23/45Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44:2715–2719

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Architecture

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Simple Lead Detector

Fluorescent ProteinFluorescent Protein

Lead Promoter

Lead Binding Protein

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Low Fluorescence

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Solution: Amplify the Signal

Fluorescent Fluorescent ProteinProtein

Lead Promoter

Amplifier

Lead Binding Protein

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Amplified Fluorescence

Low Fluorescence

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Architecture

pTet (Constitutive On)

PbrR691

Lead Promoter

PbrR691

LuxI LuxI GFP

LuxRLuxR

LuxR

AHL = Cell Signaling Molecule Made by LuxI

To other cellsTo other cells

AHL Binding Protein/Promoter

Complex

+Amplifier

AHL Producer

AHL Producer

Fluorescent

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Part Characterization

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Lead Promoter

Amplifier

1.Develop Assay to Measure AHL

2.Characterize New Parts: Lead

Promoter and Lead Binding Protein

3.Characterize Amplifier

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Experimental Steps:

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AHL = Signal

Lead Promoter

AmplifierLuxI

AHL

Output

AHL

Input

Match Output to Input to complete circuit.

PbrR691

1. AHL Assay

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How did we measure the AHL Signal?

AHL to GFP Converter (Part T9002)

1. AHL Assay

AHL GFPAHL to GFP Converter

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We can detect AHL in a Linear Fashion

0

2000

4000

6000

8000

0 M 100 pM 200 pM 500 pM 1 nM

AHL ConcentrationFluorescence

2. New Lead Parts

Lead Promoter

Lead Binding Protein

4 New Parts:

1. Lead Promoter

2. Lead Binding Protein

3. 2 Lead Binding Proteins under native promoter

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Hypothesis: Direct relationships between AHL and GFP

Result: Inverse relationship between AHL and GFP

3. Characterize Amplifier

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Inverse relationship between AHL input and GFP output

GFP output at 5 Hours

0

1000

2000

3000

4000

5000

6000

0 0.1 nM 1 nM 10 nM 100 nM 1 uM 10 uM 100 uM 1 mM

[AHL]

(GFP - control)/Cell Density

Amplifier Doesn’t Work!

Why?

Hypothesis:

AHL molecules affect

Fluorescence

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Simple Lead Detector

Fluorescent ProteinFluorescent Protein

Lead Promoter

Lead Binding Protein

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Develop AHL Assay

Characterize Amplifier

Characterize New Lead Parts

Summer Accomplishments

Community

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Tri-Stable SwitchApplying a Model

Lead DetectorPart Characterization

CommunityHighschoolersSynthetic Biology class

Brown iGEM

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John Cumbers

Alex BrodskyTayhas PalmoreGary Wessel

Multidisciplinary LabCCMBMCBMPPB

Department of Physics Division of Engineering

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