Spatial patterning and hardwired memory: engineering biomolecules as cellular input / output devices

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1 Spatial patterning and hardwired memory: engineering biomolecules as cellular input / output devices Yuki Kimura, Jack Lee, Helen Lee, Kristy Hawkins, Travis Bayer Professor Christina D. Smolke Department of Chemical Engineering California Institute of Technology March 12, 2006

2 Project goal: biological memory and spatial patterning systems Memory and spatial patterning are key cellular behaviors Design-oriented approach to construct and characterize a synthetic biological circuit that will integrate spatial addressing with signal input Selective recombination is needed for multi-dimensional hardwired memory in cells New molecules that can function as programmable input / output devices are needed for advanced cellular engineering applications

3 Project goal: biological memory and spatial patterning systems Shine image on lawn of bacteria Replica plate or recovery from a single cell (same gradient) AND gate for light and spatial address Replica plate or recovery from a single cell (w/o gradient)

4 Schematic of a biological memory system Spatial sensing RNA switch Regulation of Zinc fingers Compilation Memory register In vivo LoxP-target gene pixel plasmid biobit Master plasmid In vitro / In vivo Input signal Light-inducible promoter Cre recombinase Site-specific Spatial sensing RNA switch Zinc fingers Regulation of Output

5 A three pixel system: integrating light and spatial information input excision recombination

6 A three pixel system: integrating light and spatial information input excision recombination

7 Light memory system Spatial sensing RNA switch Regulation of Zinc fingers Compilation Memory register In vivo LoxP-target gene pixel plasmid biobit Master plasmid In vitro / In vivo Input signal Light-inducible promoter Cre recombinase Site-specific Spatial sensing RNA switch Zinc fingers Regulation of Output

8 Light regulatable site-specific recombination Red light membrane EnvZ Cph1 X OmpR OmpR P PompC Cre XCRE recombinase register X register A Levskava, AA Chevalier, JJ Tabor, ZB Simpson, LA Lavery, M Levy, EA Davidson, A Scouras, AD Ellington, EM Marcotte, CA Voigt Synthetic biology: engineering Escherichia coli to see light. Nature, 438:

9 Site-specific recombination system: Cre/LoxP recombination Cre recombinase promotes recombination at 34-bp sites, composed of two 13-bp recombinase binding elements (RBEs) arranged as inverted repeats flanking a 8-bp asymmetric crossover region which defines the directionality of the site Direct tandem repeats of result in excision (A), while inverted repeats cause inversion (B)

10 A single memory register Cre recombinase register register 1000 Fluorescence-based CRE activity assay promoter Cre recombinase gfp terminator relative GFP expression (logarithmic) GFP (IPTG) GFP+CRE (IPTG+ARA) GFP+CRE (no induction)

11 Regulatable hardwired memory Spatial sensing RNA switch Regulation of Zinc fingers Compilation Memory register In vivo LoxP-target gene pixel plasmid biobit Master plasmid In vitro / In vivo Input signal Light-inducible promoter Cre recombinase Site-specific Spatial sensing RNA switch Zinc fingers Regulation of Output

12 Regulation of s: zinc fingers Exhibit modularity in structure and function Cre recombinase Commonly arranged as covalent tandem repeats, enabling recognition of extended asymmetrical sequences zinc finger Each zinc finger is designed to target a specific register Accomplished by specific design of the DNA-binding motif to recognize a unique sequence near the LoxP site Overhang of the zinc finger will inhibit Cre activity on the adjacent memory register

13 Orthogonal zinc finger / site pairs for multi-pixel systems Cre recombinase zfp 2 zfp 3 pixel 1 pixel 2 pixel 3 pixel 2 pixel 3 pixel 1

14 Rational zinc finger design Rationally designed 6-finger zinc finger proteins to target specific nucleotide sequences using a nondegenerate recognition code table T Sera, C Uranga Rational design of artificial zinc finger proteins using a nondegenerate recognition code table. Biochemistry, 41: finger binding motif for LoxP site Position: (reverse) Finger 1: Gln-Ser-Gln-Thr Finger 2: Gln-Asp-Thr-Asn Finger 3: Thr-Asp-Arg-Asn Finger 4: Thr-Asn-Thr-Thr Finger 5: Gln-His-Glu-Thr Finger 6: Gln-Ser-Gln-Ser Berg s protein-based LoxP binding zinc finger: ZFP1: mekp ykcpecgksf sqssqlqthqrth tgekp ykcpecgksf sqsdtlqnhqrth tgekp ykcpecgksf stsdrlsnhqrth lrqkdgerp ykcpecgksf stsntlqthqrth tgekp ykcpecgksf sqshelqthqrth tgekp ykcpecgksf sqssqlsshqrthqnkk Initial zinc finger designed to bind to the entire LoxP sequence to test Cre inhibition Matching transfer functions of zinc fingers and Cre were determined to be critical for successful design

15 Spatial sensing module Spatial sensing RNA switch Regulation of Zinc fingers Compilation Memory register In vivo LoxP-target gene pixel plasmid biobit Master plasmid In vitro / In vivo Input signal Light-inducible promoter Cre recombinase Site-specific Spatial sensing RNA switch Zinc fingers Regulation of Output

16 Spatial patterning of a single concentration cut-off ON SWITCH target expression effector concentration

17 Spatial patterning of concentration ranges ON SWITCH target expression effector concentration OFF SWITCH target expression effector concentration

18 Acknowledgements The Smolke Lab Postdoctoral Researchers Kevin Hoff Graduate Researchers Andrew Babiskin Travis Bayer Chase Beisel Arwen Brown Yvonne Chen Stephanie Culler Leo D Espaux Katie Galloway Kristy Hawkins Cambrian Liu Maung Nyan Win Undergraduate Researchers Jack Lee Xin Ye Yuki Kimura Helen Lee High School Researchers Aquina Aiga Hilary Ruiz Collaborators Funding Sources The Facchini Lab (U. Calgary) The Jensen Lab (COH) The Glackin Lab (COH) Beckman Foundation Caltech Center for Biological Circuit Design (Caltech) City of Hope Cancer Center DARPA Department of Defense (BCRP) Grubstake Program (OTT Caltech) National Institutes of Health (NCI) National Institutes of Health (NIGMS) National Science Foundation (BES)