2007 Brown iGEM Team 7 undergraduates 7 grad student advisors 2 Faculty advisors 9 faculty sponsors 1/45

Brown iGEM June Update international genetically engineered machines competition 2/45

What is iGEM? Biology Engineering Standardization 3/45

Science - Nuts and Bolts Standardizing biology Systematic engineering Apply biological technology 4/45

Previous Projects Bacterial Photo Film - U. Texas (published in Nature) 5/45

Previous Projects Sepsis Treatment - Slovenia Banana/Wintergreen Smelling Cells - MIT Arsenic Water Detection - Edinburgh 5/45

Anderson, J. C. , Clarke, E. J. , Arkin, A. P. , & Voigt, C. A 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 6/45

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 7/45

by a team of undergraduates Undergraduate Teams + Imagined + Designed + Implemented by a team of undergraduates 8/45

Progress Brown’s 2nd year UTRA grants Lab space in Multi Disciplinary Lab Equipment sponsorship 9/45

Project 1: Lead Biosensor 10/45

Why do we need this? Public health concern: Lead in soil, paint, water, dust 11/45

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. 12/45

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. 13/45

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 14/45

We have spliced together biobrick parts into plasmids in E. Coli We have spliced together biobrick parts into plasmids in E. Coli. The bacteria express our genetic devices to create a lead detector. 15/45

General Design Lead Detection Lead Detection Signal Amplification Fluorescent Output 16/45

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Part 2: Signal Amplification 2 System Components Part 1: Lead Detection Part 2: Signal Amplification 18/45

Part 1: Lead Detection Lead Receptor Protein Lead Receptor Gene Always On 19/45

Activated Lead Receptor Protein Part 1: Lead Detection Lead Activated Lead Receptor Protein Lead Receptor Protein Lead Receptor Gene Always On 20/45

Part 1: Lead Detection Lead Activated Lead Receptor Protein Lead Receptor Gene Always On Signal Amplification promoter 21/45

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 22/45 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

General Design Lead Detection Lead Detection Signal Amplification Fluorescent Output 23/45

Part 2: Signal Amplification Activated Lead Receptor Protein Signal Amplification promoter 24/45

Part 2: Signal Amplification Message Activated Lead Receptor Protein Message Producer Gene Signal Amplification promoter 25/45

Part 2: Signal Amplification Message Producer Gene Signal (GFP) Message Activated Lead Receptor Protein Message Producer Gene Signal Amplification promoter 26/45

Part 2: Signal Amplification Message Producer Gene Signal (GFP) Message Activated Lead Receptor Protein Repeated activation of this promoter causes amplification of the signal. Message Producer Gene Signal Amplification promoter 27/45

3 System Components Lead Detection Signal Amplification 1. Detector Sequence 2. Leakiness Filter to Eliminate False Positives Signal Amplification 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

NO LEAD Lead Activator Message Activator Always On Transcription factors are constitutively made by the first promoter. Message Maker Filter Repressor Lead Detector Message Destroyer Filter Filter Eliminates Stray Message Molecules to Prevent False Feedback Loop Activation These proteins are poised to activate the Lead Detector promoter and Message Receiver promoter upon addition of lead. Stray Message Molecules Can Falsely Activate the Feedback Loop. Message Maker GFP Reporter Message Receiver 29/45

+ Lead Activator Feedback Activator Always On Message Maker Filter Repressor Lead turns on Detector promoter Lead Detector Message Destroyer Leakiness Filter promoter gets turned off Filter + Fluorescent Protein Output Message Maker GFP Reporter Message Receiver 30/45

Lead Activator Feedback Activator Always On Message Maker Filter Repressor 1 Lead Detector Message Destroyer 2 Filter Message Maker GFP Reporter Message Receiver 3 31/45

PbrR LuxR pTet LuxI LacI 1 Pbr aiiA 2 pLac LuxI GFP pLux 32/45 3

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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 34/45

Project 2: THE TRI-STABLE SWITCH 35/45

C A B What is Tri-stability? A tri-stable switch has three distinct and inducible states C A B 36/45

Achieving Tri-stability Input A Output A State A Input B Output B State B Input C Output C State C 37/45

The Architecture Arabinose RFP pBad/Ara tetR lacI IPTG CFP pLac araC Tetracycline YFP pTet lacI araC 38/45

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 40/45

Timeline Now: August: End of lab work November: Jamboree at MIT 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 41/45

Why Brown? Innovators Entrepreneurs A great place for new ideas! 42/45

The Future Educate others about iGEM and synthetic biology Synthetic Biology Course Offering in Fall 07! Led by Prof. Gary Wessel 43/45

Special Thanks To: Office of the Dean of the College Office of the President The Atlantic Philanthropies The Center for Computational and Molecular Biology Department of Physics Engineering Department Department of Molecular Biology, Cell Biology, and Biochemistry Department of Molecular Pharmacology, Physiology, and Biotechnology The Multi Disciplinary Lab Pfizer Labnet Nanodrop 44/45

Thank you for listening! Questions? 45/45