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Design of Digital Logic by Genetic Regulatory Networks Ron Weiss Department of Electrical Engineering Princeton University Computing Beyond Silicon Summer School, Caltech, Summer 2002
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E. coli Diffusing signal Programming Cell Communities proteins Program cells to perform various tasks using: Intra-cellular circuits –Digital & analog components Inter-cellular communication –Control outgoing signals, process incoming signals
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Programmed Cell Applications analyte source Analyte source detection Reporter rings Pattern formation Biomedical –combinatorial gene regulation with few inputs; tissue engineering Environmental sensing and effecting –recognize and respond to complex environmental conditions Engineered crops –toggle switches control expression of growth hormones, pesticides Cellular-scale fabrication –cellular robots that manufacture complex scaffolds
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Outline In-vivo logic circuits Intercellular communications Signal processing and analog circuits Programming cell aggregates
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A Genetic Circuit Building Block Digital Inverter Threshold Detector Amplifier Delay
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Logic Circuits based on Inverters Proteins are the wires/signals Promoter + decay implement the gates NAND gate is a universal logic element: –any (finite) digital circuit can be built! X Y R1R1 Z R1R1 R1R1 X Y Z = gene NANDNOT
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Why Digital? We know how to program with it –Signal restoration + modularity = robust complex circuits Cells do it –Phage λ cI repressor: Lysis or Lysogeny? [Ptashne, A Genetic Switch, 1992] –Circuit simulation of phage λ [McAdams & Shapiro, Science, 1995] Also working on combining analog & digital circuitry
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BioCircuit Computer-Aided Design SPICE BioSPICE steady statedynamics intercellular BioSPICE: a prototype biocircuit CAD tool – simulates protein and chemical concentrations – intracellular circuits, intercellular communication – single cells, small cell aggregates
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Genetic Circuit Elements input mRNA ribosome promoter output mRNA ribosome operator translation transcription RNAp RBS
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Modeling a Biochemical Inverter input output repressor promoter
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A BioSPICE Inverter Simulation input output repressor promoter
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“Proof of Concept” Circuits Work in BioSPICE simulations [Weiss, Homsy, Nagpal, 1998] They work in vivo –Flip-flop [Gardner & Collins, 2000] –Ring oscillator [Elowitz & Leibler, 2000] However, cells are very complex environments –Current modeling techniques poorly predict behavior time (x100 sec) [A][A] [C][C] [B][B] B _S_S _R_R A _[R]_[R] [B][B] _[S]_[S] [A][A] RS-Latch (“flip-flop”)Ring oscillator
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The IMPLIES Gate Inducers that inactivate repressors: –IPTG (Isopropylthio-ß-galactoside) Lac repressor –aTc (Anhydrotetracycline) Tet repressor Use as a logical Implies gate: (NOT R) OR I operatorpromoter gene RNA P active repressor operator promoter gene RNA P inactive repressor inducer no transcription transcription Repressor Inducer Output
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The Toggle Switch [Gardner & Collins, 2000] pIKE = lac/tet pTAK = lac/cIts
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Actual Behavior of Toggle Switch [Gardner & Collins, 2000] promoter protein coding sequence
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The Ring Oscillator [Elowitz, Leibler 2000]
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Example of Oscillation
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Evaluation of the Ring Oscillator Reliable long-term oscillation doesn’t work yet: Will matching gates help? Need to better understand noise Need better models for circuit design [Elowitz & Leibler, 2000]
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A Ring Oscillator with Mismatched Inverters A = original cI/ λ P(R) B = repressor binding 3X weaker C = transcription 2X stronger
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Device Physics in Steady State Transfer curve: gain (flat,steep,flat) adequate noise margins [input] “gain” 01 [output] Curve can be achieved with certain dna-binding proteins Inverters with these properties can be used to build complex circuits “Ideal” inverter
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Measuring a Transfer Curve Construct a circuit that allows: –Control and observation of input protein levels –Simultaneous observation of resulting output levels “drive” geneoutput gene R YFPCFP inverter Also, need to normalize CFP vs YFP
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Flow Cytometry (FACS)
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Drive Input Levels by Varying Inducer 0 100 1000 IPTG YFP lacI [high] 0 (Off) P(lac) P(lacIq) lacI P(lacIq) YFP P(lac) IPTG IPTG (uM) promoter protein coding sequence
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Also use for CFP/YFP calibration Controlling Input Levels
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Cell Population Behavior Red = pPROLAR Rest = pINV-102 with IPTG (0.1 to 1000 uM)
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CFP: a Weak Fluorescent Protein Induction of CFP expression IPTG Fluorescence
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Measuring a Transfer Curve for lacI/p(lac) aTc YFP lacI CFP tetR [high] 0 (Off) P(LtetO-1) P(R) P(lac) measure TC tetR P(R) P(Ltet-O1) aTc YFP P(lac) lacICFP
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Transfer Curve Data Points 01011010 1 ng/ml aTc undefined 10 ng/ml aTc100 ng/ml aTc
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lacI/p(lac) Transfer Curve aTc YFP lacI CFP tetR [high] 0 (Off) P(LtetO-1) P(R) P(lac) gain = 4.72
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Evaluating the Transfer Curve Noise margins:Gain / Signal restoration: high gain * note: graphing vs. aTc (i.e. transfer curve of 2 gates)
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Transfer Curve of Implies YFP lacI aTc IPTG tetR [high]
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The Cellular Gate Library Add the cI/ P(R) Inverter OR1OR1OR2OR2 structural gene P(R-O12) cI is a highly efficient repressor cooperative binding IPTG YFP cI CFP lacI [high] 0 (Off) P(R) P(lac) Use lacI/p(lac) as driver high gain cI bound to DNA
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Initial Transfer Curve for cI/ P(R) lacI P(lacIq) P(lac) IPTG YFP P(R) cICFP
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Recall Inverter Components input mRNA ribosome promoter output mRNA ribosome operator translation transcription RNAp RBS
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Functional Composition of an Inverter “clean” signaldigital inversionscale inputinvert signal translation 01 01 01 ++= “gain” 01 ++= cooperative binding transcriptioninversion input mRNA input protein bound operators output mRNA
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Genetic Process Engineering I: Reducing Ribosome Binding Site Efficiency RBS translation start Orig: ATTAAAGAGGAGAAATTAAGCATG strong RBS-1: TCACACAGGAAACCGGTTCGATG RBS-2: TCACACAGGAAAGGCCTCGATG RBS-3: TCACACAGGACGGCCGGATG weak translation stage Inversion
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Experimental Results for cI/ P(R) Inverter with Modified RBS
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Genetic Process Engineering II: Mutating the P(R) operator BioSPICE Simulation orig: TACCTCTGGCGGTGATA mut4: TACATCTGGCGGTGATA mut5: TACATATGGCGGTGATA mut6 TACAGATGGCGGTGATA OR1OR1 cooperative binding
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Experimental Results for Mutating P(R)
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Genetic Process Engineering Genetic modifications required to make circuit work Need to understand “device physics” of gates –enables construction of complex circuits modify RBS mutate operator RBS #1: modify RBS #2: mutate operator
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Self-perfecting Genetic Circuits [Arnold, Yokobayashi, Weiss] optical micrograph of the μFACS device Use directed evolution to optimize circuits Screening criteria based on transfer curve Initial results are promising Lab-on-a-chip: μFACS [Quake]
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Molecular Evolution of the Circuit
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Prediction of Circuit Behavior = ? Output signal Input signal Can the behavior of a complex circuit be predicted using only the behavior of its parts?
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Prediction of Circuit Behavior preliminary results YFP aTc # cells tetR P(bla) P(tet) aTc cIP(lac) lacICFP YFP P(R)
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