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Genetically Engineered Neuronal Networks: Genetically Engineered Neuronal Networks: Th F Th F The Fast and the Furious! The Fast and the Furious! t t d th F d th F i i ! !

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

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

Synthetic Biology Bottom Synthetic Biology Bottom- -Up Up Approach Approach Systems Modules Devices Physical

Outline Outline Neurons Neurons M d li Modeling Differentiation Patterning Toggle Action Potentials

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

How are Impulses Transmitted? How are Impulses Transmitted? • Ion gradients • Electrical charge El t i l h • Voltage-gated channels de = -k e e dt di 1 = k i 1 = -k i i1 1 dt di 2 = -k i + a i i2 2 i1 1 dt dm = -k m + a e + a i m e i2 2 dt 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 N1 N1 Input ! 1: Input ! 1: Input ! 1: Input ! 1: Input ! 1: Input ! 1: Input ! 2: Input ! 2: Input ! 2: Input ! 2: Input ! 2: Input ! 2: N1 ! N1 ! N1 ! N1 ! N1 ! N1 ! N2 ! N2 ! N2 ! N2 ! N2 ! N2 ! Dopamine Dopamine Dopamine Dopamine Output Output Output Output Output Output p p p Output Output Output Output Output Output p p p GABA GABA GABA GABA GABA GABA GABA GABA GABA GABA GABA GABA Glycine Glycine Glycine Glycine Glycine Glycine y y y 0 0 0 0 0 0 0 0 0 Hold Hold Hold Hold Hold Hold Hold Hold Hold Hold Hold Hold Glycine Glycine 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 0 - - - - - - - 1 1 1 1 1 1 1 1 1 " P Exogenous Exogenous Inhibitor Inhibitor [GABA] [GABA] GABA GABA Dopamine Dopamine D D i i N2 N2 Excitatory Excitatory Inhibitory Inhibitory Inhibitory Inhibitory

Stem Cells Stem Cells ! Neuronal Neuronal P Precursors recursors Dox TRE TRE N Ngn1 1 EYFP EYFP mKate K t Ub Ubc Bl Bla Ubc rtTA 2A

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 Nurr1 GABA or Glycine Cav3.1 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 y y • Computationally efficient Pacemakers: • 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 I 1 P 1 N 1 E P 2 N 2 I 2 E E Neither N1 nor N2 respond to P 1 sequential inhibitory inputs ti l i hibit i t N 1

Functional Functional Bistability Bistability • Thresholds and timing for functional circuit E P 1 P 2 2 N 1 N N 2

Toggle Switch: Spatial Design Toggle Switch: Spatial Design N1 •Dopamine ! Receptor •GABAergic i •Glycine Receptor Pacemaker ! Cells •Constant ! Output •Dopaminergic N2 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 of Gold Surfaces Microfabrication of Gold Surfaces Cell Adhesion Patterning • Encouragement of directed E t f di t d • Gold layer on glass slide axonal growth upwards • Cover with thiol and through apex dendrimers dendrimers • Effectively breaks the • Grow cells on dendrimers 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 divided P N1 N2 P • Gold deposited using thermal evaporator • Connections large enough C ti l h to let a few axons through

Adhesion to Gold Surfaces Adhesion to Gold Surfaces

Pacemaker Genetic Constructs Pacemaker Genetic Constructs Ubc rtTA LacI/Krab IRES Puro 2A IPTG Dox Hef1a/LacO Mash1 Nurr1 Ubc Hyg yg 2A TRE Cav3.1 Ubc Bleo TRE HCN2 Ubc Bla

N2 Genetic Constructs N2 Genetic Constructs Ubc rtTA LacI/Krab IRES Puro 2A IPTG Hef1A/LacO H f1A/L O Mash1 M h1 GFP GFP Ub Ubc H Hyg Dox TRE TRE GlyT2 GlyT2 VIAAT VIAAT Ubc Ubc Bla Bla 2A TRE D5R Ubc Bleo TRE GABAR Gephyrin Ubc Neo 2A

N1 Genetic Constructs N1 Genetic Constructs Ubc rtTA LacI/Krab IRES Puro 2A IPTG IPTG Dox Hef1A/LacO Mash1 GFP Ubc Hyg TRE Lbx1 GLRA1 D5R Ubc Bla 2A 2A

Negative ! control: Experiment: Experiment: No ! agonist With ! agonist 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?