1. In vitro biochemical circuits Leader: Erik Winfree co-leader: Jongmin Kim 1.The synthetic biology problem 2.The experimental system we are investigating 3.A general problem it motivates 4.A specific problem to tackle

2. In vitro biochemical circuits Leader: Erik Winfree co-leader: Jongmin Kim 1.The synthetic biology problem Reductionism: system behavior from component characteristics The complexity gap Synthesis of in vitro biochemical circuits 2.The experimental system we are investigating 3.A general problem it motivates 4.A specific problem to tackle

3. In vitro biochemical circuits Leader: Erik Winfree co-leader: Jongmin Kim 1.The synthetic biology problem 2.The experimental system we are investigating Circuits of rationally-designed transcriptional switches 3.A general problem it motivates 4.A specific problem to tackle ? RNase RNA DNA RNAP promoter DA R A I R [R] [A] tot [I] tot

4. 1.The synthetic biology problem 2.The experimental system we are investigating 3.A general problem it motivates There are many subspecies and side reactions. How do we obtain a simplified model for analysis? 4.A specific problem to tackle By RNA polymerase ON OFF In vitro biochemical circuits Leader: Erik Winfree co-leader: Jongmin Kim By RNase

5. In vitro biochemical circuits Leader: Erik Winfree co-leader: Jongmin Kim 1.The synthetic biology problem 2.The experimental system we are investigating 3.A general problem it motivates 4.A specific problem to tackle Phase space analysis of simple circuits: a bistable switch and a ring oscillator 1 0 0 1 10 e.g. “cloud size”

6. Mass action chemical kinetics

7. An adjustable transcriptional switch

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

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. Sequence design TCATGGAACTACAACAGGCAACTAATACGACTCACTATAGGGAGAAGCAACGATACGGTCTAGAGTCACTAAGAGTAATACAGAACTGACAAAGTCAGAAA GCTGAGTGATATCCC TC TTCG TTGCTATG CCAGATCTCAGTGATTCT CATTAT GTCTTGACTG TTTC AGTCTTTGTGTTCCT AGTACCTTGATGTT GTCCGTTGATTAT Promoter 85 27 hairpin AGCAACGATACGGTCTAGAGTCACTAAGAGTAATACAGAA AAA GGGAGA CTGAC GTCAG A A A 6 827 Signal

12. Components RNAP RNase H RNase R D 12 D 21 A2A2 A1A1 ATTGAGGTAAGAAAGGTAAGGATAATACGACTCACTATAGGGAGAAACAAAGAACGAACGACACTAATGAACTACTACTACACACTAATACTGACAAAGTCAGAAA CTAATGAACTACTACTACACACTAATACGACTCACTATAGGGAGAAGGAGAGGCGAAGATTGAGGTAAGAAAGGTAAGGATAATACTGACAAAGTCAGAAA TATTAGTGTGTAGTAGTAGTTCATTAGTGTCGTTC TATTATCCTTACCTTTCTTACCTCAATCTTCGCCT TTTCTGACTTTGTCAGTATTATCC TT ACC TTT C TT ACCTCAATCTTCGCCTCTCCTTCTCCCTATAGTGAGTCG TTTC TGACTTTGTCAGTATTAGTGTGTAGTAGTAGTTCATTAGTGTCGTTCG TTCTTTGTTTCTCCCTATAGTGAGTCG

13. Transition curve – DNA inhibitor T7 RNAP RNase H(1U) RNase R(200nM) D 21 =100nM A=500nM Sw21 Inh1 Inh2 add DNA Inhibitor 2 A tot dI1 I2

14. Transition curve – RNA inhibitor T7 RNAP RNase H(0.7U) RNase R(150nM) D 13 =0-60nM D 21 =80nM A=400nM A tot Inhibitor 2 Inhibitor 1 Sw21 Sw13 Inh2 Inh1 I2 I1

15. Fluorescence OFF ON High signal Low signal

16. Bistable switch Inh 2 Inh 1 Sw 21 Sw 12

17. Bistable switch Sw 12 ON Sw 21 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. Inhibitor interacting with Switch/Enzyme complex D A I RNAP I + RDA -> RD + AI I A D RNAP

21. Abortive transcripts (Messiness #1) D RNAP A D A I R + DA RDA -> R + DA + I 60, I 45, I 14,I 8

22. RNase R needs to clean up I 8, I 14 RNase R Rr + I n RrI n -> Rr

23. Activator crosstalk D 21 A2A2 A2A2 D 21 + A 2 -> D 21 A 2

24. Nicked at -12/-13 has no crosstalk I2I2 A 1 or A 2 D 21 Stoichiometric amounts of activator Transcription level (%) spnonspnon 1x0x1x2x3x1x0x1x2x3x 1002824 10010 119 T7 RNAP D 21 =100nM, 500nM D 21 +A 1 D 21 D 21 +A 2

25. Incomplete degradation by RNaseH (Messiness #2) I 45 A hp RNase H A I RhAI -> Rh + A + I n + hp

26. RNase H can keep going I 45 A RNase H I 27 A RNase H I 14 A RNase H Rh + AI n RhAI n -> Rh + AI m I 27 A I 14 A RNase H

27. Lots of truncated RNA products I2 I2 hairpin ? R(0nM)R(100nM)R(200nM)R(400nM) D 21 =30nM A=150nM T7 RNAP RNase H(1.5U) RNase R 60120 60180 12060 180 Sw21 Inh2 sI2

28. Activator-activator or Inhibitor- inhibitor complex II 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) D 21 =30nM A=150nM T7 RNAP RNase H(1.5U) RNase R 60120 60180 12060 180 Sw21 Inh2 Extended I2 complex I2

31. Enzyme life-time RNAP R -> ø

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

33. I2 level is stable (up to ~6hr) R(0nM)R(100nM)R(200nM)R(400nM) D 21 =30nM A=150nM T7 RNAP RNase H(1.5U) RNase R 60120 60180 12060 180 Sw21 Inh2 I2

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

35. DNA bands are stable R(0nM)R(100nM)R(200nM)R(400nM) D 21 =30nM A=150nM T7 RNAP RNase H(1.5U) RNase R 60120 60180 12060 180 Sw21 Inh2 DNA sense DNA temp BH-A

36. Initial burst D RNAP A D A I 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 + I n AI n DA + I n DAI n D + AI n R + DA RDA -> R + DA + I n R + DAI 1 n RDAI 1 n -> R + DAI 1 n + I 2 n’ R + D RD -> R + D + I n Rh + AI n RhAI n -> Rh + AI m (+ hp) Rr + I n RrI n -> 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