2007 Brown iGEM Team

•7 undergraduates

•7 grad student advisors

•2 Faculty advisors

•9 faculty sponsors

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Brown iGEMinternational genetically engineered machines competition

June Update

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What is iGEM?

• Biology

• Engineering

• Standardization

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Science - Nuts and Bolts

•Standardizing biology

•Systematic engineering

•Apply biological technology

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Previous Projects

•Bacterial Photo Film - U. Texas (published in Nature)

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Previous Projects

•Sepsis Treatment - Slovenia

•Banana/Wintergreen Smelling Cells - MIT

•Arsenic Water Detection - Edinburgh

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Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2005) Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria, Journal of Molecular Biology

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Aerobic Conditions

Low Cell Density

>0.02% Arabinose

OFF

Hypoxia

High Cell Density

<0.02% Arabinose

ON

Inv

induction

INVASION

Anderson, J. C., Clarke, E. J., Arkin, A. P., & Voigt, C. A. (2005) Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria, Journal of Molecular Biology

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Undergraduate Teams

+ Imagined+ Designed

+ Implemented

by a team of undergraduates

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Progress

•Brown’s 2nd year

•UTRA grants

•Lab space in Multi Disciplinary Lab

•Equipment sponsorship

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Project 1: Lead Biosensor

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Why do we need this?Public health concern: Lead in soil, paint, water, dust

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Why do we need this?Public health concern: Lead in soil, paint, water, dust

Lead Poisoning is often caused by ingesting contaminated drinking water, or soil. It can cause neurological and gastrointestinal disorders, especially among children.

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Why do we need this?Public health concern: Lead in soil, paint, water, dust

Lead Poisoning is often caused by ingesting contaminated drinking water, or soil. It can cause neurological and gastrointestinal disorders, especially among children.

The legal limit of lead in drinking water is 15 parts per billion.

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Why do we need this?

Current ways of testing for lead either require expensive chemical lab analysis or involve inaccurate home kits.

So why do we want a biosensor?

- Cheap

- Sensitive

- Quick

- Specific

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We have spliced together biobrick parts into plasmids in E. Coli.

The bacteria express our genetic devices to create a lead detector.

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Lead Detection

Signal Amplification

Fluorescent Output

Lead

General Design

Lead Detection

Signal Amplification

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2 System Components

Part 1: Lead Detection

Part 2: Signal Amplification

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Always On

Lead Receptor

Gene

Lead Receptor Protein

Part 1: Lead Detection

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Always On

Lead Receptor

Gene

Lead Receptor Protein

Lead

Activated Lead

Receptor Protein

Part 1: Lead Detection

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Activated Lead

Receptor Protein

Always On

Lead Receptor

Gene

Lead Receptor Protein

Lead

Activated Lead

Receptor Protein

Signal Amplification

promoter

Part 1: Lead Detection

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Part 1: Lead Detection This is sensitive ONLY to lead; other metals will not activate it.

Chen 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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Lead Detection

Signal Amplification

Fluorescent Output

Lead

General Design

Lead Detection

Signal Amplification

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Activated Lead

Receptor Protein

Signal Amplification

promoter

Part 2: Signal Amplification

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Activated Lead

Receptor Protein

Signal Amplification

promoter

Part 2: Signal Amplification

Message Producer

Gene

Message

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Activated Lead

Receptor Protein

Signal Amplification

promoter

Part 2: Signal Amplification

Message Producer

Gene

MessageMessage Producer

Gene

Signal (GFP)

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Activated Lead

Receptor Protein

Signal Amplification

promoter

Part 2: Signal Amplification

Message Producer

Gene

MessageMessage Producer

Gene

Signal (GFP)

Repeated activation of this promoter causes amplification of the signal.

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3 System Components

Lead Detection

Signal Amplification

1. Detector Sequence

2. Leakiness Filter to Eliminate False Positives

3. Positive Feedback Loop for Amplification

We’ve added a “Leakiness Filter” as an additional component to the system. This gives our circuit one more level of complexity.

However, the black box diagram is generally the same. 28/45

Always On

Lead Activator

Message Activator

Lead Detector

Message Maker

Filter Repressor

FilterMessage Destroyer

Message Receiver

Message Maker

GFP Reporter

Stray Message Molecules Can Falsely Activate the Feedback

Loop.

Filter Eliminates Stray Message Molecules to

Prevent False Feedback Loop

Activation

NO LEADTranscription factors

are constitutively made by the first promoter.

These proteins are poised to activate the

Lead Detector promoter and Message Receiver promoter upon addition

of lead.29/45

Always On

Lead Activator

Feedback Activator

Lead Detector

Message Maker

Filter Repressor

FilterMessage Destroyer

Message Receiver

Message Maker

GFP Reporter

+Fluorescent

Protein Output

Lead turns on Detector promoter

Leakiness Filter promoter gets turned off

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Always On

Lead Activator

Feedback Activator

Lead Detector

Message Maker

Filter Repressor

FilterMessage Destroyer

Message Receiver

Message Maker

GFP Reporter

1

2

3 31/45

pTetPbrR LuxR

PbrLuxI LacI

pLacaiiA

pLuxLuxI GFP

1

2

3 32/45

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How this project advances science:

• Characterization of Existing Parts

• Adding New Parts and Devices– Lead Promoter and Transcription Factor– Amplifier Device

• Generalize to future biosensors– Arsenic, Cadmium, Mercury, Zinc

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THE TRI-STABLESWITCH

Project 2:

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What is Tri-stability?• A tri-stable

switch has three distinct and inducible states

ABC

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Achieving Tri-stability

State A

State C

State B

Output A

Output B

Output C

Input A

Input CInput B

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pBad/Ara tetR

tetR

lacI

lacI

pLac araC

araCpTet

RFP

CFP

YFP

The ArchitectureArabinose

IPTG

Tetracycline

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Characterization• Characterization is an essential aspect of iGEM

• It is a step towards standardization - giving others all the details needed to use the part. 39/45

Uses

• Differentiation of stem cells

• Turn on/off three different proteins in cell

• Cellular logic• Tissue Engineering

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Timeline• Now:

– Start cloning– PCR lead promoter– Clone tri-stable switch– Characterize parts– Test systems– Send back to the Registry

• August: End of lab work• November: Jamboree at MIT

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Why Brown?

•Innovators

•Entrepreneurs

•A great place for new ideas!

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The Future

•Educate others about iGEM and synthetic biology

•Synthetic Biology Course Offering in Fall 07! Led by Prof. Gary Wessel

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Office of the Dean of the CollegeOffice of the President

The Atlantic PhilanthropiesThe Center for Computational and Molecular Biology

Department of PhysicsEngineering Department

Department of Molecular Biology, Cell Biology, and Biochemistry

Department of Molecular Pharmacology, Physiology, and Biotechnology

The Multi Disciplinary LabPfizerLabnet

Nanodrop

Special Thanks To:

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Thank you for listening!

Questions?45/45