Meet the Brown Team Ahmad Rana Michael Chang Ahmad Ran-a-Gel MC - PowerPoint PPT Presentation

Meet the Brown Team Ahmad Rana Michael Chang Ahmad Ran-a-Gel MC Mastermix Stephanie Cheung Stephylococcus William Allen Ashley Kim Will Allenquot Ashley Kimwipe Minoo Ramanathan Brown iGEM Indu Voruganti Minoo-Prep The Inducer Elias Scheer Flora Ko E. coli Scheer Nasal Flora 2009 Brown iGEM 1

Brown iGEM A Synthetic Approach to Treating Nasal Allergies 2009 Brown iGEM 2

Brown iGEM 2009 2009 Brown iGEM 3

Allergic Rhinitis $11.2 billion American Academy of Allergy Asthma and Immunology 2009 Brown iGEM 4

Project Overview Photo Credits: Paesan et al, 1999 (top); Janice Carr, CDC (bottom) 2009 Brown iGEM 5

The Allergic Response 2009 Brown iGEM 6

The Allergic Response Allergen B cell Histamine Mast Cell IgE Antibodies Plasma Cell 2009 Brown iGEM 7

The Allergic Response Mast Cell Histamine Nerve Histamine Receptors Receptor Vasodilation Bloodstream 2009 Brown iGEM 8

Current Antihistamine Treatment Mast Cell Histamine Antihistamine Nerve Histamine Receptors Receptor Bloodstream 2009 Brown iGEM 9

Allergene Schematic Histamine Histamine Sensor Binding Protein and Signaling Staphylococcus Quorum Sensor epidermidis Purpose: To sequester histamine in the event of an allergic attack 2009 Brown iGEM 10

Histamine Binding Protein rEV131 Rhipicephalus appendiculatus Paesen, G.C., et al. "Tick Histamine-Binding Proteins…” Molecular Cell , 1999 , 3 , 661-671. 2009 Brown iGEM 11

Allergene Schematic 4. Histamine Sequestration 3. Secretion 1. Histamine Sensing P OmpC OmpR Sec rEV131 2. Signaling / transcription 2009 Brown iGEM 12

Allergene Schematic Histamine Sensor and Signaling Purpose: To sense elevated histamine levels in an allergic attack and signal a response 2009 Brown iGEM 13

Mutating the Receptor: RBP The Receptor We altered an existing prokaryotic receptor, ribose binding protein to make our histamine sensor. Native Ribose Binding Protein with Ribose 2009 Brown iGEM 14

Mutating the Receptor: RBP mRBP Receptor Design Modified RBP (mRBP) Native RBP with Histamine with Ribose Poly-Alanine RBP with Ribose Rosettacommons.org 2009 Brown iGEM 15

Mutating the Receptor: RBP Native RBP with Ribose Histamine Receptor with Histamine 2009 Brown iGEM 16

Mutating the Receptor: RBP 2009 Brown iGEM 17

Mutating the Receptor: RBP Histamine Receptor scores nearly as well as RBP in silico ! Ligand Total Packing Buried Protein Ligand Stat w/o Total Packing Unsat. Total H Interface Energy Ligand Score Stat Polars Bonds Exposure Histamine Receptor -1075.378 0.663 0.62 53 292 -13.02 0.964 RBP -1061.185 0.668 0.655 61 291 -14.616 1 (2DRI) S"ll in the process of tes"ng in vivo / vitro 2009 Brown iGEM 18

Allergene Schematic 1. Histamine Sensing P OmpC OmpR Sec rEV131 2009 Brown iGEM 19

The Histamine Sensor Linking Ligand Detection to a Cascade Trg (normally receives Ligand-bound RBP) fused to EnvZ (intracellular kinase) Histamine Receptor H EnvZ Periplasmic Domain Trg Binds His Osmosensor Receptor Chemotaxis Intracellular Intracellular Domain Signaling Kinase 2009 Brown iGEM 20

The Histamine Sensor EnvZ Signaling Cascade 1 Histamine Receptor 1. Ligand-bound receptor binds to Trg domain. Trg 4 2. EnvZ phosphorylates 2 transcription factor OmpR . 3 P 3. OmpR- P activates OmpC OmpC EnvZ OmpR promoter. Sec rEV131 4. Activation of OmpC promoter leads to gene transcription (rEV131) 2009 Brown iGEM 21

Testing the Cascade Fluorescence Assay Trg-EnvZ + OmpC-RFP Reporter Cassette OmpC rEV131 RFP Just Trg-EnvZ Just OmpC-RFP This assay will also be used to test histamine binding to our mutated receptors. 2009 Brown iGEM 22

Allergene Schematic P OmpC OmpR Sec rEV131 2. Signaling / transcription 2009 Brown iGEM 23

Allergene Schematic Staphylococcus epidermidis Purpose: To serve as the delivery vehicle of our genetic construct 2009 Brown iGEM 24

Why choose S. epidermidis? E. coli S. aureus S. epidermidis Secrete proteins? Secrete proteins? Secrete proteins? Nasal flora? Nasal flora? Nasal flora? Non-pathogenic? Non-pathogenic? Present in ~30% Present in ~90% Good for modeling of people’s nasal of people’s nasal vestibule. vestibule. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2003, p. 18–23 Vol. 69, No. 1 2009 Brown iGEM 25

Microorganismal Treatment Platform Allergene uses a microorganism as the base of operations for treatment within a host’s body. This has emerged in years past at iGEM (BACTOBlood, BACTOKidney) Advantage of our design We use a species native to the nasal cavity 2009 Brown iGEM 26

Allergene Schematic rEV131 must be released from the cell to sequester extracellular histamine 3. Secretion P OmpC OmpR Sec rEV131 S. epidermidis 2009 Brown iGEM 27

Secretion Testing Secretion Ligated secretion peptide to GFP using BioBrick Assembly Reporter Cassette Signal peptide causes GFP secretion OmpC rEV131 Sec Pep GFP 2009 Brown iGEM 28

Allergene Schematic Quorum Sensor Purpose: To maintain a safe population of bacteria 2009 Brown iGEM 29

Quorum Sensor Agr Signal Transduction Cascade S. Epidermidis Biofilm Auto-inducing peptide (AIP) secreted by population of cells AIP AIP AIP AIP AIP pAgr CCDB GFP Biofilm 2009 Brown iGEM 30

Quorum Sensor Testing the Quorum Sensor In our test construct, pAgr promotes GFP to test its efficacy when a quorum is reached. Quorum reached, S. epidermidis low pAgr induced, GFP density, pAgr not fluoresces induced, no GFP fluorescence 2009 Brown iGEM 31

Allergene Schematic 4. Histamine Sequestration 3. Secretion 1. Histamine Sensing P OmpC OmpR Sec rEV131 Quorum Sensor 2. Signaling / transcription S. epidermidis 2009 Brown iGEM 12

Acknowledgements • Dr. Gary Wessel • Dr. Guido Paesan • Adrian Reich • Dr. Loren Looger • Diana Donovan • Dr. Masayori Inouye • John Szymanski • Dr. Luciano Marraffini • PRIMO Lab • Dr. Reinholb Bruckner • Barnea Lab • Center for Computational Molecular Biology • Brown UTRA Program • Division of Engineering • Dr. Sorin Istrail • Division of Biology and Medicine • Adella Francis • ThermoFisher Scientific • Aaron Glieberman • Department of Molecular Biology, • Neil Parikh, Kate Jacobs, Rima Shah Cell Biology, and Biochemistry • John Cumbers • Brown Multidisciplinary Laboratory 2009 Brown iGEM 33

Brown iGEM Ahmad Rana Michael Chang Ahmad Ran-a-Gel MC Mastermix Stephanie Cheung Stephylococcus William Allen Ashley Kim Will Allenquot Ashley Kimwipe Minoo Ramanathan Brown iGEM Indu Voruganti Minoo-Prep The Inducer Elias Scheer Flora Ko E. coli Scheer Nasal Flora 2009 Brown iGEM 34

Summary • Curing allergies with a self-regulating histamine-sequestering drug factory in your nose! • Histamine-binding protein, rEV131 , from ticks, outcompetes histamine binding to receptors • Used Staphylococcus epidermidis as chassis • Computationally designed a novel histamine receptor • Created a signaling fusion protein • Designed safety mechanism to control population size • Added secretion tag to secrete histamine-binding protein 2009 Brown iGEM 35