Transcript lacl
Project Overview Landing Pads Divide-by two Circuit •Genetic toggle switch switchable by one input •Essential computing component Modeling •General, useful tool for all synthetic biologists •A systematic, Biobrick-compatible approach to put plasmid constructs onto the chromosome Fabrication •Characterize system •Construct gene operons in •Determine robustness plasmid form and assemble into the whole system and predict ways to improve performance Insert DBT constructs into landing pad and cross into chromosome Divide-By-Two Circuit Idealized Divide-By-Two Circuit Output (normalized) input Time • The same input toggles the system between both states (on and off) • Frequency of the output is half of the frequency of the input • Output can be any gene expression DBT Circuit: Single Input Toggle Switch σ54 NR1 GFP lacp araB gfp lacl glnG cI glnKp rpoN LacI araBp RFP cl nifHp σ54 cl cIp nifA nifA lacl rfp How It Works NR1 GFP lacl lacp gfp glnG cI lacl rfp glnKp rpoN araBp nifHp cl cIp nifA How It Works σ54 NR1 GFP araB lacl lacp gfp glnG cI lacl rfp glnKp rpoN araBp nifHp σ54 cl cIp nifA How It Works σ54 NR1 GFP lacp lacl gfp glnG cI lacl rfp glnKp rpoN LacI araBp nifHp σ54 cl cIp nifA How It Works σ54 lacp lacl gfp glnG cI lacl rfp glnKp rpoN LacI araBp nifHp σ54 cl cIp nifA How It Works lacp lacl gfp glnG cI glnKp rpoN RFP araBp nifHp cl cIp nifA nifA lacl rfp How It Works σ54 araB lacp lacl gfp glnG cI glnKp rpoN RFP araBp nifHp σ54 cl cIp nifA nifA lacl rfp How It Works σ54 lacp lacl gfp glnG cI glnKp rpoN RFP araBp cl nifHp σ54 cl cIp nifA nifA lacl rfp How It Works σ54 lacp lacl gfp glnG cI lacl rfp glnKp rpoN araBp cl nifHp σ54 cl cIp nifA How It Works NR1 GFP lacl lacp gfp glnG cI lacl rfp glnKp rpoN araBp nifHp cl cIp nifA Modeling QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Basic ODE’s VglnKp ( 54 ) nsl ( NR1) ngl VcIp K cl ncl d (lacI ) ncl lacI (lacI ) ngl nsl n n cl n dt ( K sl ( 54 ) sl )( K gl ( NR1) gl ) K cl cI VNifHp ( 54 )nsc ( NifA)nnc Vlacp Klc nlc d (cI ) nlc cI (cI ) nsc nsc nnc nnc nlc dt ( Kbd ( 54 ) )( K nc ( NifA) ) Klc lacI •Hill function and degradation •No Basal production Steady State Analysis • Under which conditions will the system toggle and which conditions will it not? • If it can toggle, how “easy” is it to achieve this? • How robust is the system? Reducing Variable Dimensions σ54 NR1 GFP lacp araB gfp lacl glnG cI glnKp rpoN LacI araBp RFP cl nifHp σ54 cl cIp nifA nifA lacl rfp Steady State Equations lacI cI VcIp K cl •Two variables and 8 parameters ncl ncl lacI ( K cl cI ) ncl Vlacp K lc nlc nlc cI ( K lc lacI ) nlc •Generally the parameters are either unknown and/or vary over a range •Symmetric vs unsymmetric Existence of Multiple Steady States NO INPUT 3 steady 1 steady states: state: 2 stable, stable 1 unstable cI Phase plane: one threesteady steadystate states lacI Toggle System Needs Two Steady States With INPUT Two stable steady toggle Single steady state:states: no toggle canpossible happen Input added: Inputtoggle added:between no toggle two states cI GFP RFP lacI What Parameter Ranges Yield Toggle Activity? • Only a narrow range of parameter values give toggle behavior • How robust is the system? – Not very • This will be addressed later So how do you toggle? Vary parameters assuming… No toggle possible Toggle possible •How does input affect the system? •Is a toggle easily achieved? System responds differently to varying input levels Sufficient Low input Excess input input Response of lacI and cI: insufficient sufficient excess input input input concentration Steady state 1 Transient response Ideal! Quasi-steady state QuasiSteady state 2 1 bifurcation ??? time The Nature of Quasi-Steady State Original system: Two stable steady states ONE quasiSteady state Output 1 Back to original steady states Output 1 Output 2 High s54 level Excess input added S54 degrades Output 2 Quasi-Steady State Heavily Favors One Output End: Output 1 Start: Output NOT 1 TOGGLE BEHAVIOR!!! 2 Output 1 Output 1 Output 2 High s54 level Excess input added S54 degrades Output 2 Dynamic Modeling of the DBT • State transition takes place 1. Rapidly increases 2. Dips back down 3. Slowly rises to dominant level Concentration (molecules) • “Overthrowing” side: lacI cI Time (s) Dynamic Modeling of the DBT Concentration lacI cI Time Potential Problem Spots A: Input promoter must be very quiet A B: Cross-talk between the two values must be low C: lacp and cIp side parameter values must be relatively close B C • Device predicted to tend to have lacdominant side more stable • Can switch into lacdominant side, but can’t switch out Concentration (molecules) • Large difference in the parameters between sides Concentration (molecules) Unbalanced, Gardner-based Simulations: lacI: (blue) cI: (gold) rfp: (red) gfp: (green) Time (s) lacI: (blue) cI: (gold) rfp: (red) gfp: (green) Time (s) Stochastic Modeling •Mass-Action Model and Gillespie Method Tuning the Device with IPTG and Temperature • Attenuate Lac with IPTG • Attenuate cI with heat • Calibrate device by varying both IPTG and temperature • Current research focusing on if and where “sweet-spot” is located Clamping the DBT • Regardless of input pulse length/decay rate, only one change-of-states occurs Divide-By-Two Fabrication Operons of the DBT Circuit • Initial construction requires five plasmids, each carrying one operon of the circuit • Our final design will have fewer plasmids, as we will place some on the chromosome and combine others cIp-nifA-lacI-RFP BBa_I720004 BBa_I720005 lacp-GFP-glnG-cI lacp-GFP-glnG-cI (cont) • Sequencing and characterization failed – BioBrick had bad DNA (wrong lacp sequence) – Actually needed repressible, rather than constitutive, promoter – Sequencing results verified plasmid had all the genes, though BBa_I720006 BBa_I720002 PCR: NCM 77 PCR: K. Pneumoniae genome BBa_I720003 BBa_I720000 BBa_I720001 • Characterization: – transformed plasmid into rpoN mutants: growth regardless of arabinose input – Also transformed rpoN alone: still have growth – Too noisy; need a single copy – Back-up plan: tetO-rpoN DBT Operons in Landing Pads Synthetic Operon Landing Pad Used Leucine BioBrick Landing Pad BBa_I720007 Arabinose BioBrick Landing Pad glnK Landing Pad DBT Operons in Landing Pads BioBrick Landing Pad Built so that ANYONE can insert ANY BioBrick onto the Chromosome of E. Coli! BioBrick Landing Pad: Goals of Project • General Landing Pad Goal: Aid in insertion of constructs onto the chromosome • Develop a general method for constructing landing pads that: • Have BioBrick compatible restriction sites • Allow easy phenotypic screening • Limit noise • Allow nesting of sequential landing pads BioBrick Landing Pad BioBrick Landing Pad BioBrick Landing Pad: Homologous Regions Arabinose Homologous Regions Homologous Recombination of Arabinose Landing Pad BioBrick Landing Pad: Nesting Landing Pads Benefits of Nested Landing Pads • Allow for more constructs to be inserted onto the chromosome at the same chromosomal location • Different drug resistance gene is used: Only one type of screening is required BioBrick Landing Pad: Nesting Landing Pads New Landing Pad Criteria • Homologous regions from previous drug resistance gene (ChlorR) • Different drug resistance gene (KanR) BioBrick Landing Pad: Nested Landing Pads Nested Product: Chloramphenicol Landing Pad nested in Arabinose Landing Pad BioBrick Landing Pad: Fabrication Progress Landing Pads 250 bp Arabinose Landing Pad 500 bp Arabinose Landing Pad 1000 bp Arabinose Landing Pad 250 bp Maltose Landing Pad 500 bp Maltose Landing Pad 1000 bp Maltose Landing Pad Status Completed Completed Completed In Progress In Progress In Progress Registry Number BBa_I720008 BBa_I720009 BBa_I7200010 N/A N/A N/A * Nesting to be tried in the near future as well! • Prof. Alex Ninfa • Domitilla DelVecchio • Prof. Peter Woolf • Dong Eun Chang Questions? Unbalanced System: Bifurcation favors one state • In a more realistic unbalanced system, the quasi-SS leads to one of the two steady states • One steady state is favored over the other Quasi-Steady State Bifurcates with Sigma54 level (Balanced System) • If the system is balanced, as sigma54 degrades past threshold both steady states are equally accessible Stable SS Unstable SS Quasi SS Sigma54 concentration Low input: no sigma54 presence after response Time