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

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

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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

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SLIDE 1

2009 Brown iGEM 1

Meet the Brown Team

William Allen

Will Allenquot

Michael Chang

MC Mastermix

Stephanie Cheung

Stephylococcus

Ashley Kim

Ashley Kimwipe

Flora Ko

Nasal Flora

Elias Scheer

  • E. coli Scheer

Minoo Ramanathan

Minoo-Prep

Indu Voruganti

The Inducer

Brown iGEM

Ahmad Rana

Ahmad Ran-a-Gel

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SLIDE 2

2009 Brown iGEM 2

Brown iGEM

A Synthetic Approach to Treating Nasal Allergies

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SLIDE 3

2009 Brown iGEM 3

Brown iGEM 2009

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SLIDE 4

2009 Brown iGEM 4

Allergic Rhinitis

American Academy of Allergy Asthma and Immunology

$11.2 billion

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SLIDE 5

2009 Brown iGEM 5

Project Overview

Photo Credits: Paesan et al, 1999 (top); Janice Carr, CDC (bottom)

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SLIDE 6

2009 Brown iGEM 6

The Allergic Response

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SLIDE 7

2009 Brown iGEM 7

The Allergic Response

B cell Allergen Plasma Cell IgE Antibodies Mast Cell Histamine

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SLIDE 8

2009 Brown iGEM 8

The Allergic Response

Vasodilation

Mast Cell Histamine Receptor Histamine

Nerve Receptors Bloodstream

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SLIDE 9

2009 Brown iGEM 9

Current Antihistamine Treatment

Mast Cell Histamine Receptor Histamine Antihistamine

Bloodstream Nerve Receptors

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SLIDE 10

2009 Brown iGEM 10

Allergene Schematic

Histamine Sensor and Signaling Staphylococcus epidermidis Quorum Sensor Histamine Binding Protein

Purpose: To sequester histamine in the event of an allergic attack

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SLIDE 11

2009 Brown iGEM 11

Histamine Binding Protein

rEV131

Paesen, G.C., et al. "Tick Histamine-Binding Proteins…” Molecular Cell, 1999 , 3, 661-671.

Rhipicephalus appendiculatus

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SLIDE 12

2009 Brown iGEM 12

Allergene Schematic

OmpR

P

  • 4. Histamine

Sequestration

  • 1. Histamine Sensing
  • 2. Signaling / transcription
  • 3. Secretion

Sec rEV131 OmpC

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SLIDE 13

2009 Brown iGEM 13

Allergene Schematic

Purpose: To sense elevated histamine levels in an allergic attack and signal a response

Histamine Sensor and Signaling

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SLIDE 14

2009 Brown iGEM 14

Mutating the Receptor: RBP

Native Ribose Binding Protein with Ribose

The Receptor

We altered an existing prokaryotic receptor, ribose binding protein to make our histamine sensor.

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SLIDE 15

2009 Brown iGEM 15

Mutating the Receptor: RBP

Poly-Alanine RBP with Ribose Native RBP with Ribose Modified RBP (mRBP) with Histamine

mRBP Receptor Design

Rosettacommons.org

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SLIDE 16

2009 Brown iGEM 16

Mutating the Receptor: RBP

Histamine Receptor with Histamine Native RBP with Ribose

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SLIDE 17

2009 Brown iGEM 17

Mutating the Receptor: RBP

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SLIDE 18

2009 Brown iGEM 18

Mutating the Receptor: RBP

Total Score Packing Stat Packing Stat w/o Ligand Total Buried Unsat. Polars Total H Bonds Ligand Protein Interface Energy Ligand Exposure Histamine Receptor -1075.378 0.663 0.62 53 292

  • 13.02

0.964 RBP (2DRI)

  • 1061.185

0.668 0.655 61 291

  • 14.616

1

Histamine Receptor scores nearly as well as RBP in silico!

S"ll in the process of tes"ng in vivo/vitro

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SLIDE 19

2009 Brown iGEM 19

Allergene Schematic

OmpR

P

  • 1. Histamine Sensing

Sec rEV131 OmpC

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SLIDE 20

2009 Brown iGEM 20

The Histamine Sensor

Linking Ligand Detection to a Cascade

Trg (normally receives Ligand-bound RBP) fused to EnvZ (intracellular kinase) Periplasmic Domain Intracellular Domain Chemotaxis Signaling Trg EnvZ Osmosensor Binds His Receptor Intracellular Kinase H Histamine Receptor

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SLIDE 21

2009 Brown iGEM 21

The Histamine Sensor

EnvZ Signaling Cascade

  • 1. Ligand-bound receptor

binds to Trg domain.

  • 2. EnvZ phosphorylates

transcription factor OmpR.

  • 3. OmpR- P activates OmpC

promoter.

  • 4. Activation of OmpC

promoter leads to gene transcription (rEV131) Trg EnvZ

OmpR

P

Sec rEV131 OmpC 1 2 3 4 Histamine Receptor

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SLIDE 22

2009 Brown iGEM 22

Testing the Cascade

Trg-EnvZ + OmpC-RFP

Fluorescence Assay

This assay will also be used to test histamine binding to our mutated receptors. RFP OmpC Reporter Cassette Just Trg-EnvZ Just OmpC-RFP rEV131

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SLIDE 23

2009 Brown iGEM 23

Allergene Schematic

OmpR

P

  • 2. Signaling / transcription

Sec rEV131 OmpC

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SLIDE 24

2009 Brown iGEM 24

Allergene Schematic

Staphylococcus epidermidis

Purpose: To serve as the delivery vehicle of our genetic construct

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2009 Brown iGEM 25

Why choose S. epidermidis?

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2003, p. 18–23 Vol. 69, No. 1

Secrete proteins? Nasal flora? Secrete proteins? Nasal flora? Non-pathogenic? Secrete proteins? Nasal flora? Non-pathogenic?

  • E. coli
  • S. aureus
  • S. epidermidis

Present in ~90%

  • f people’s nasal

vestibule. Present in ~30%

  • f people’s nasal

vestibule. Good for modeling

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SLIDE 26

2009 Brown iGEM 26

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

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SLIDE 27

2009 Brown iGEM 27

Allergene Schematic

rEV131 must be released from the cell to sequester extracellular histamine OmpR

P

Sec rEV131 OmpC

  • 3. Secretion
  • S. epidermidis
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SLIDE 28

2009 Brown iGEM 28

Secretion

Testing Secretion

Signal peptide causes GFP secretion

Ligated secretion peptide to GFP using BioBrick Assembly

GFP OmpC rEV131 Reporter Cassette Sec Pep

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SLIDE 29

2009 Brown iGEM 29

Allergene Schematic

Quorum Sensor

Purpose: To maintain a safe population

  • f bacteria
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SLIDE 30

2009 Brown iGEM 30

Quorum Sensor

Agr Signal Transduction Cascade

  • S. Epidermidis Biofilm

Auto-inducing peptide (AIP) secreted by population of cells

AIP AIP AIP AIP AIP

pAgr GFP CCDB Biofilm

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SLIDE 31

In our test construct, pAgr promotes GFP to test its efficacy when a quorum is reached.

2009 Brown iGEM 31

Quorum Sensor

Quorum reached, pAgr induced, GFP fluoresces

  • S. epidermidis low

density, pAgr not induced, no GFP fluorescence

Testing the Quorum Sensor

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SLIDE 32

2009 Brown iGEM 12

Allergene Schematic

OmpR

P

  • 4. Histamine

Sequestration

  • 1. Histamine Sensing
  • 2. Signaling / transcription
  • 3. Secretion

Sec rEV131 OmpC

Quorum Sensor

  • S. epidermidis
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SLIDE 33

2009 Brown iGEM 33

Acknowledgements

  • Dr. Gary Wessel
  • Adrian Reich
  • Diana Donovan
  • John Szymanski
  • PRIMO Lab
  • Barnea Lab
  • Brown UTRA Program
  • Dr. Sorin Istrail
  • Adella Francis
  • Aaron Glieberman
  • Neil Parikh, Kate Jacobs, Rima Shah
  • John Cumbers
  • Dr. Guido Paesan
  • Dr. Loren Looger
  • Dr. Masayori Inouye
  • Dr. Luciano Marraffini
  • Dr. Reinholb Bruckner
  • Center for Computational Molecular

Biology

  • Division of Engineering
  • Division of Biology and Medicine
  • ThermoFisher Scientific
  • Department of Molecular Biology,

Cell Biology, and Biochemistry

  • Brown Multidisciplinary Laboratory
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SLIDE 34

2009 Brown iGEM 34

Brown iGEM

William Allen

Will Allenquot

Michael Chang

MC Mastermix

Stephanie Cheung

Stephylococcus

Ashley Kim

Ashley Kimwipe

Flora Ko

Nasal Flora

Elias Scheer

  • E. coli Scheer

Minoo Ramanathan

Minoo-Prep

Indu Voruganti

The Inducer

Brown iGEM

Ahmad Rana

Ahmad Ran-a-Gel

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SLIDE 35

2009 Brown iGEM 35

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