Th F Th F The Fast and the Furious! The Fast and the Furious! t - - PowerPoint PPT Presentation

Genetically Engineered Neuronal Networks: Genetically Engineered Neuronal Networks:

Th F t d th F i ! Th F t d th F i ! The Fast and the Furious! The Fast and the Furious!

Why Neurons? Why Neurons?

http://www.sciencedaily.com/images/2008/01/080109091102.jpg http://www.makeadifference.ie/brain%20circuit.jpg http://student.britannica.com/elementary/art-88968/The-boxer-Muhammad-Ali-and-the-actor-Michael-J-Fox

Medical Applications Medical Applications

http://www.absolutechiropractor.com/Images/cns.jpg http://www.chm.bris.ac.uk/webprojects2006/Cowlishaw/mech%20synaptic%20transmission.htm

Synthetic Biology Bottom Synthetic Biology Bottom-

• Up

Up Approach Approach

Systems Modules Devices Physical

Outline Outline

Neurons M d li Neurons Modeling Differentiation Patterning Toggle Action Potentials

Neurons Neurons

http://www.nia.nih.gov/

How are Impulses Transmitted? How are Impulses Transmitted?

• Ion gradients

El t i l h

• Electrical charge
• Voltage-gated channels

= -k e de dt

e

di

1 = k i 1

i1 1

dt = -k i di2 dt = -k i + a i

i2 2 i1 1

dm dt = -k m + a e + a i

m e i2 2

http://www.chm.bris.ac.uk/webprojects2006/Cowlishaw/300px-Action-potential.png p p j p p p

Excitation vs. Inhibition Excitation vs. Inhibition

http://download.biologia-en-internet.com/images/biocom/campbell-reece.jpg

Our Toggle Switch Our Toggle Switch

Input!1: Input!1:

GABA GABA

Input!2: Input!2:

Glycine Glycine

N1! N1! Output Output N2! N2! Output Output

N1 N1

Input!1: Input!1:

GABA GABA

Input!2: Input!2:

Glycine Glycine

N1! N1! Output Output N2! N2! Output Output Input!1: Input!1:

GABA GABA

Input!2: Input!2:

Glycine Glycine

N1! N1! Output Output N2! N2! Output Output

Dopamine Dopamine

GABA GABA

y

p p Hold Hold Hold Hold 1 1 1 1 1 1 1

Glycine Glycine

GABA GABA

y

p p Hold Hold Hold Hold 1 1 1 1 1 1 1

GABA GABA

y

p p Hold Hold Hold Hold 1 1 1 1 1 1 1

Dopamine Dopamine

1 1 1 "

• Exogenous

Exogenous

1 1 1

• 1

1 1

• P

D i D i Inhibitor Inhibitor [GABA] [GABA] GABA GABA

Excitatory Excitatory Inhibitory Inhibitory

Dopamine Dopamine N2 N2

Inhibitory Inhibitory

Stem Cells Stem Cells ! Neuronal Neuronal P Precursors recursors

Dox

EYFP K t Ub Bl TRE N 1

2A

Ubc rtTA EYFP mKate Ubc Bla TRE Ngn1

System Design System Design

N1

• Dopamine Receptor
• GABAergic
• Glycine Receptor

Pacemaker Cells

• Constant Spikes
• Dopaminergic

N2

• Dopamine Receptor
• GABA Receptor
• GABA Receptor
• Glycinergic

System Design Details System Design Details

DRD5 Lbx1 GLRA1 HCN2

• r

Cav3.1 Nurr1 GABA Glycine GABAR GLYT2 DRD5

Modeling the Toggle Modeling the Toggle

NEURON: NEURON: well-suited to problems with experimental data, especially networks and cells involving complex anatomical properties

IntFire4:

• Artificial spiking cell
• Can both be excitatory and inhibitory

Pacemakers: y y

• Computationally efficient
• Receive stream of electrical stimuli,

resulting in activation of neurons N1 & N2 Neurons N1/N2: Neurons N1/N2:

• Excited by pacemaker cells
• Cross-inhibit

Implementation of the Model Implementation of the Model

Connections First Attempt

I1 P1 P2 E N1 N2 I2

E Neither N1 nor N2 respond to ti l i hibit i t P1 E sequential inhibitory inputs N1

Functional Functional Bistability Bistability

• Thresholds and timing for functional circuit

E P1 P2 N

1

N

2

N

2

Toggle Switch: Spatial Design Toggle Switch: Spatial Design

N1

• Dopamine!Receptor

i

• GABAergic
• Glycine Receptor

N2 Pacemaker!Cells

• Constant!Output
• Dopaminergic

Dopamine!Receptor

• GABA!Receptor
• Glycinergic

Specification: Specification:

• Asymmetric transmission of ‘signals’
• Physical separation of each ‘cluster’ of neurons

Physical separation of each cluster of neurons

• Ability to replicate experiments with consistent results

Microfabrication Microfabrication of Gold Surfaces

• f Gold Surfaces

Cell Adhesion Patterning E t f di t d

• Gold layer on glass slide
• Cover with thiol and

dendrimers

• Encouragement of directed

axonal growth upwards through apex dendrimers

• Grow cells on dendrimers
• Effectively breaks the

symmetry of the networks

http://ieeexplore.ieee.org/stamp/stamp.jsp?arnumber=01279475 http://www.nature.com/nphys/journal/vaop/ncurrent/full/nphys1099.html

Geometric Design Geometric Design

• Pacemaker cells can be

divided

P N1 N2 P

divided

• Gold deposited using

thermal evaporator C ti l h

• Connections large enough

to let a few axons through

Adhesion to Gold Surfaces Adhesion to Gold Surfaces

Pacemaker Genetic Constructs Pacemaker Genetic Constructs

2A

Ubc rtTA LacI/Krab IRES Puro Hef1a/LacO Mash1 Ubc Hyg

IPTG Dox

Nurr1

2A

yg TRE Cav3.1 Ubc Bleo TRE HCN2 Ubc Bla

N2 Genetic Constructs N2 Genetic Constructs

Ubc rtTA LacI/Krab IRES Puro

2A

H f1A/L O M h1 GFP Ub H

IPTG

TRE GlyT2 VIAAT Ubc Bla

Dox

Hef1A/LacO Mash1 GFP Ubc Hyg

2A

TRE GlyT2 VIAAT Ubc Bla TRE D5R Ubc Bleo

2A

TRE GABAR Gephyrin Ubc Neo

N1 Genetic Constructs N1 Genetic Constructs

2A

Ubc rtTA LacI/Krab IRES Puro

IPTG Dox

Hef1A/LacO Mash1 GFP Ubc Hyg

IPTG

TRE Lbx1 GLRA1 D5R Ubc Bla

2A 2A

Negative!control: No!agonist Experiment: With!agonist Experiment: With!agonist Normalized background Normalized!background

What We’ve Accomplished What We’ve Accomplished

• Genetically engineered multi-phase differentiation into

neurons

• Successfully modeled and analyzed toggle switch
• Patterned cells on surfaces
• Dopamine agonist induced action potential response
• Dopamine agonist induced action potential response

Future Work Future Work

• Finish neuronal toggle switch

– Construction of remaining plasmids for toggle switch p – Viral production and stem cell infection of remaining toggle genes

• Full circuit testing of toggle

– Integration neuronal populations Integration neuronal populations – Testing alternative layouts for learning circuit

• Application of Toggle: Learning

Modeling of learning paradigms – Modeling of learning paradigms – Proof of principle testing of learning circuit with exogenous neurotransmitters Full circuit testing of learning circuit – Full circuit testing of learning circuit

• Potential applications for re-growth of brain tissue
• Interface with other biological sensors and actuators

The Princeton iGEM Team The Princeton iGEM Team

Our team: Virender Kanwal Navin Duseja Andrew Gordon Hamza Masood Our team: Virender Kanwal , Navin Duseja, Andrew Gordon, Hamza Masood, Molly Herring, Lena Phalen, Katia Sherman, David Glass, Eric Finkelstein, Caroline Richardson, Evan Kelly. Our Instructors: Ron Weiss Cil Purnick Caroline DeHart Patrick Guye Noah Our Instructors: Ron Weiss, Cil Purnick, Caroline DeHart, Patrick Guye, Noah Davidsohn, Saurabh Gupta Our Lab Manager: Steve Firsing Thanks to: Assaf Rotem, Ken Norman, Oliver Graudejus, Bernd Kuhn, PRISM, , , j , , , ELE, CIEE, MOL, NSF EMT, CHE, Provost

Questions? Questions?