In vitro biochemical circuits

1. In vitro biochemical circuits The synthetic biology problem The experimental system we are investigating A general problem it motivates A specific problem to tackle Leader: Erik Winfree co-leader: Jongmin Kim

2. In vitro biochemical circuits • The synthetic biology problem Reductionism: system behavior from component characteristics The complexity gap Synthesis of in vitro biochemical circuits • The experimental system we are investigating • A general problem it motivates • A specific problem to tackle Leader: Erik Winfree co-leader: Jongmin Kim

3. RNAP RNA ? RNase DNA In vitro biochemical circuits • The synthetic biology problem • The experimental system we are investigating Circuits of rationally-designed transcriptional switches • A general problem it motivates • A specific problem to tackle [R] Leader: Erik Winfree co-leader: Jongmin Kim R [I]tot DA [A]tot promoter I A R

4. In vitro biochemical circuits • The synthetic biology problem • The experimental system we are investigating • A general problem it motivates There are many subspecies and side reactions. How do we obtain a simplified model for analysis? • A specific problem to tackle OFF ON Leader: Erik Winfree co-leader: Jongmin Kim ON OFF By RNA polymerase By RNase

5. 0 1 1 0 0 1 In vitro biochemical circuits • The synthetic biology problem • The experimental system we are investigating • A general problem it motivates • A specific problem to tackle Phase space analysis of simple circuits: a bistable switch and a ring oscillator Leader: Erik Winfree co-leader: Jongmin Kim e.g. “cloud size”

6. Mass action chemical kinetics

7. An adjustable transcriptional switch

8. OFF ON ON OFF By RNA polymerase By RNase Networks of transcriptional switches

9. Michaelis-Menten reactions Michaelis-Menten reactions lead to competition for - RNA polymerase by DNA templates - RNase by RNA products Can have interesting consequences like Winner-take-all network

10. Experimental system

11. 8 6 27 8 5 hairpin 27 Signal Sequence design TCATGGAACTACAACAGGCAACTAATACGACTCACTATAGGGAGAAGCAACGATACGGTCTAGAGTCACTAAGAGTAATACAGAACTGACAAAGTCAGAAA GTGTTCCT AGTACCTTGATGTT GTCCGTTGATTAT GCTGAGTGATATCCC TC TTCG TTGCTATG CCAGATCTCAGTGATTCT CATTAT GTCTTGACTG TTTC AGTCTTT Promoter A A A GGGAGA GTCAG CTGAC AGCAACGATACGGTCTAGAGTCACTAAGAGTAATACAGAA AAA

12. Components D12 ATTGAGGTAAGAAAGGTAAGGATAATACGACTCACTATAGGGAGAAACAAAGAACGAACGACACTAATGAACTACTACTACACACTAATACTGACAAAGTCAGAAA TTTC TGACTTTGTCAGTATTAGTGTGTAGTAGTAGTTCATTAGTGTCGTTCG TTCTTTGTTTCTCCCTATAGTGAGTCG TATTATCCTTACCTTTCTTACCTCAATCTTCGCCT A2 D21 CTAATGAACTACTACTACACACTAATACGACTCACTATAGGGAGAAGGAGAGGCGAAGATTGAGGTAAGAAAGGTAAGGATAATACTGACAAAGTCAGAAA TATTAGTGTGTAGTAGTAGTTCATTAGTGTCGTTC TTTCTGACTTTGTCAGTATTATCC TT ACC TTT C TT ACCTCAATCTTCGCCTCTCCTTCTCCCTATAGTGAGTCG A1 RNAP RNase H RNase R

13. Transition curve – DNA inhibitor T7 RNAP RNase H(1U) RNase R(200nM) I2 D21=100nM A=500nM Inhibitor 2 Inh2 add DNA Sw21 dI1 Inh1 Atot

14. Transition curve – RNA inhibitor T7 RNAP RNase H(0.7U) RNase R(150nM) I2 D13=0-60nM D21=80nM A=400nM Inhibitor 2 Sw21 Inhibitor 1 Inh2 Inh1 Atot I1 Sw13

15. Fluorescence OFF High signal ON Low signal

16. Bistable switch Sw21 Inh1 Inh2 Sw12

17. Bistable switch Sw21 ON Sw12 ON

18. Summary • Need better quantitative understanding • make a better system • understand how messy system works Cells have misfolded, mutated species all the time Neural networks have distributed architecture

19. Possible complications

20. I I A A D D Inhibitor interacting with Switch/Enzyme complex RNAP RNAP I + RDA -> RD + AI

21. I A A D D Abortive transcripts (Messiness #1) RNAP RNAP R + DA <-> RDA -> R + DA + I60, I45, I14 ,I8

22. RNase R needs to clean up RNase R I8, I14 RNase R Rr + In <-> RrIn -> Rr

23. A2 D21 D21 Activator crosstalk A2 D21 + A2 -> D21A2

24. Nicked at -12/-13 has no crosstalk D21+A1 D21+A2 D21 T7 RNAP D21=100nM, 500nM D21 I2 A1 or A2 Stoichiometric amounts of activator Transcription level (%)

25. I A Incomplete degradation by RNaseH (Messiness #2) RNase H I45 hp RNase H A RhAI -> Rh + A + In + hp

26. RNase H can keep going RNase H I45 Rh + AIn <-> RhAIn -> Rh + AIm A RNase H I27 I27 RNase H A A RNase H RNase H I14 I14 A A

27. Lots of truncated RNA products R(0nM) R(100nM) R(200nM) R(400nM) T7 RNAP RNase H(1.5U) RNase R 60 120 180 60 120 180 60 120 180 60 120 180 D21=30nM A=150nM I2 Inh2 sI2 Sw21 I2 hairpin ?

28. I I Activator-activator or Inhibitor-inhibitor complex I I I + I -> II

29. RNA extension by RNAP RNAP I I’ RNAP R + I -> RI -> R + I’

30. Extended RNA species R(0nM) R(100nM) R(200nM) R(400nM) T7 RNAP RNase H(1.5U) RNase R 60 120 180 60 120 180 60 120 180 60 120 180 Extended I2 complex D21=30nM A=150nM I2 Inh2 Sw21

31. Enzyme life-time RNAP R -> ø

32. I A A D D NTP/buffer exhaustion RNAP CTP ATP GTP UTP RNAP RDA + 60NTP -> R+ DA + I

33. I2 level is stable (up to ~6hr) R(0nM) R(100nM) R(200nM) R(400nM) T7 RNAP RNase H(1.5U) RNase R 60 120 180 60 120 180 60 120 180 60 120 180 D21=30nM A=150nM I2 Inh2 Sw21

34. A RNase degrading DNA RNase H RNase H Rh + A -> RhA -> Rh

35. DNA bands are stable R(0nM) R(100nM) R(200nM) R(400nM) T7 RNAP RNase H(1.5U) RNase R 60 120 180 60 120 180 60 120 180 60 120 180 D21=30nM A=150nM DNA sense DNA temp Inh2 BH-A Sw21

36. I A A D D Initial burst RNAP RNAP RDA -> R + DA + I k(t)

37. Model choice (basic) D + A <-> DA A + I <-> AI DA + I <-> DAI -> D + AI R + DA <-> RDA -> R + DA + I R + D <-> RD -> R + D + I Rh + AI <-> RhAI -> Rh + A Rr + I <-> RrI -> Rr

38. Model choice (with messiness) D + A <-> DA A + In <-> AIn DA + In <-> DAIn <-> D + AIn R + DA <-> RDA -> R + DA + In R + DAI1n <-> RDAI1n -> R + DAI1n + I2n’ R + D <-> RD -> R + D + In Rh + AIn <-> RhAIn -> Rh + AIm (+ hp) Rr + In <-> RrIn -> Rr

39. Questions • Bistable circuit phase diagram • Oscillator circuit phase diagram • Bistable circuit model reduction • Oscillator circuit model reduction • Transcription switch input/output model reduction