Project Overview

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

3. 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

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

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

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

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

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

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

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

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

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

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

14. Modeling

15. Basic ODE’s • Hill function and degradation • No Basal production

16. 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?

17. NR1 NR1 σ54 GFP GFP lacp lacp araB lacl lacl gfp gfp glnG glnG cI cI glnKp glnKp rpoN rpoN LacI LacI araBp araBp RFP RFP cl cl nifHp nifHp cl cl nifA nifA lacl lacl rfp rfp cIp cIp nifA nifA σ54 Reducing Variable Dimensions

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

19. 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

20. Toggle System Needs Two Steady States With INPUT Two stable steady states: toggle possible Single steady state: no toggle can happen Input added: toggle between two states Input added: no toggle GFP cI RFP lacI

21. 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

22. So how do you toggle? Vary parameters assuming… Toggle possible No toggle possible • How does input affect the system? • Is a toggle easily achieved?

23. 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

24. 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 Output 2 High s54 level Excess input added S54 degrades

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

26. 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)

27. Dynamic Modeling of the DBT lacI Concentration cI Time

28. 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

29. 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)

30. Stochastic Modeling • Mass-Action Model and Gillespie Method

31. 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

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

33. Divide-By-Two Fabrication

34. 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

35. cIp-nifA-lacI-RFP BBa_I720004 BBa_I720005

36. lacp-GFP-glnG-cI

37. 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

38. BBa_I720002 PCR: NCM 77 BBa_I720003 PCR: K. Pneumoniae genome

39. 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 BBa_I720000 BBa_I720001

40. DBT Operons in Landing Pads BBa_I720007

41. DBT Operons in Landing Pads

42. BioBrick Landing Pad Built so that ANYONE can insert ANY BioBrick onto the Chromosome of E. Coli!

43. 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

44. BioBrick Landing Pad

45. BioBrick Landing Pad

46. BioBrick Landing Pad: Homologous Regions Arabinose Homologous Regions Homologous Recombination of Arabinose Landing Pad

47. 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

48. BioBrick Landing Pad: Nesting Landing Pads • New Landing Pad Criteria • Homologous regions from previous drug resistance gene (ChlorR) • Different drug resistance gene (KanR)

49. BioBrick Landing Pad: Nested Landing Pads Nested Product: Chloramphenicol Landing Pad nested in Arabinose Landing Pad

50. BioBrick Landing Pad: Fabrication Progress * Nesting to be tried in the near future as well!