Project Overview Landing Pads Divide-by two Circuit Modeling General, useful tool for all synthetic biologists A systematic, Biobrick-compatible approach to put plasmid constructs onto the chromosome Divide-by two Circuit Genetic toggle switch switchable by one input Essential computing component Modeling Characterize system Determine robustness and predict ways to improve performance Fabrication Construct gene operons in plasmid form and assemble into the whole system Insert DBT constructs into landing pad and cross into chromosome

Divide-By-Two Circuit Idealized Divide-By-Two Circuit 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 input Output (normalized) Time

DBT Circuit: Single Input Toggle Switch σ54 NR1 GFP lacp araB lacl gfp glnG cI glnKp rpoN LacI araBp RFP cl nifHp cl nifA lacl rfp cIp σ54 nifA

How It Works lacp lacl rpoN nifHp cl rfp NR1 GFP gfp glnG cI glnKp araBp nifHp cl nifA lacl rfp cIp

How It Works lacp lacl rpoN nifHp cl rfp σ54 NR1 GFP araB gfp glnG cI glnKp rpoN araBp nifHp cl nifA lacl rfp cIp σ54

How It Works lacp lacl rpoN nifHp cl rfp σ54 NR1 GFP gfp glnG cI glnKp LacI araBp nifHp cl nifA lacl rfp cIp σ54

How It Works lacp lacl rpoN nifHp cl rfp σ54 gfp glnG cI glnKp LacI araBp nifHp cl nifA lacl rfp cIp σ54

How It Works lacp lacl rpoN nifHp cl rfp gfp glnG cI glnKp araBp RFP nifA lacl rfp cIp nifA

How It Works lacp lacl rpoN nifHp cl rfp σ54 araB gfp glnG cI glnKp araBp RFP nifHp cl nifA lacl rfp cIp σ54 nifA

How It Works lacp lacl rpoN nifHp cl rfp σ54 gfp glnG cI glnKp araBp nifA lacl rfp cIp σ54 nifA

How It Works lacp lacl rpoN nifHp cl rfp σ54 gfp glnG cI glnKp araBp nifA lacl rfp cIp σ54

How It Works lacp lacl rpoN nifHp cl rfp NR1 GFP gfp glnG cI glnKp araBp nifHp cl nifA lacl rfp cIp

Modeling

Basic ODE’s 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 lacp gfp LacI lacl cI cl araBp rpoN glnG NR1 nifA nifHp glnKp cIp rfp GFP RFP lacp gfp LacI lacl cI cl araBp araB rpoN glnG σ54 NR1 nifA nifHp glnKp cIp rfp GFP RFP

Steady State Equations Two variables and 8 parameters Generally the parameters are either unknown and/or vary over a range Symmetric vs unsymmetric

Existence of Multiple Steady States NO INPUT 3 steady states: 2 stable, 1 unstable 1 steady state: stable Phase plane: three steady states Phase plane: one steady state cI lacI

Toggle System Needs Two Steady States With INPUT Two stable steady states: toggle possible Single steady state: no toggle can happen Input added: no toggle Input added: toggle between two states GFP cI 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 Toggle No toggle possible assuming… Toggle possible No toggle possible How does input affect the system? Is a toggle easily achieved?

System responds differently to varying input levels Sufficient input Low input Excess input Ideal! Steady state 1 Response of lacI and cI: excess input Response of lacI and cI: sufficient input Response of lacI and cI: insufficient input Transient response concentration Quasi-steady state Quasi- Steady state Steady state 1 Steady state 2 bifurcation ??? time

The Nature of Quasi-Steady State Back to original steady states Original system: Two stable steady states ONE quasi- Steady state Output 1 Output 1 Output 2 High s54 level Output 2 Excess input added S54 degrades

Quasi-Steady State Heavily Favors One Output End: Output 1 Start: Output 2 Start: Output 1 NOT TOGGLE BEHAVIOR!!! Output 1 Output 1 Output 2 High s54 level Output 2 Excess input added S54 degrades

Dynamic Modeling of the DBT State transition takes place “Overthrowing” side: Rapidly increases Dips back down Slowly rises to dominant level lacI Concentration (molecules) cI Time (s) 26

Dynamic Modeling of the DBT lacI Concentration cI Time 27

Potential Problem Spots A: Input promoter must be very quiet B: Cross-talk between the two values must be low C: lacp and cIp side parameter values must be relatively close C B A 28

Concentration (molecules) Concentration (molecules) Unbalanced, Gardner-based Simulations: lacI: (blue) cI: (gold) rfp: (red) gfp: (green) Large difference in the parameters between sides Device predicted to tend to have lac-dominant side more stable Can switch into lac-dominant side, but can’t switch out Concentration (molecules) Time (s) lacI: (blue) cI: (gold) rfp: (red) gfp: (green) Concentration (molecules) Time (s) 29

Stochastic Modeling Mass-Action Model and Gillespie Method 30

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 31

Clamping the DBT Regardless of input pulse length/decay rate, only one change-of-states occurs 32

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 35

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 BBa_I720003 PCR: K. Pneumoniae genome 38

Also transformed rpoN alone: still have growth 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 39

DBT Operons in Landing Pads Synthetic Operon Landing Pad Used Leucine BioBrick Landing Pad Arabinose BioBrick Landing Pad glnK Landing Pad BBa_I720007

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 * Nesting to be tried in the near future as well!

Questions? THANK YOU!!! Prof. Alex Ninfa Prof. Peter Woolf Domitilla DelVecchio 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

Low input: no sigma54 presence after response Sigma54 concentration Time