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

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

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

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

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 e.g. “cloud size”

Mass action chemical kinetics

An adjustable transcriptional switch

Networks of transcriptional switches By RNA polymerase ON OFF By RNase

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

Experimental system

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

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

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

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

Fluorescence OFF ON High signal Low signal

Bistable switch Inh 2 Inh 1 Sw 21 Sw 12

Bistable switch Sw 12 ON Sw 21 ON

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

Possible complications

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

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

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

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

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

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

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

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 Sw21 Inh2 sI2

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

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

Extended RNA species R(0nM)R(100nM)R(200nM)R(400nM) D 21 =30nM A=150nM T7 RNAP RNase H(1.5U) RNase R Sw21 Inh2 Extended I2 complex I2

Enzyme life-time RNAP R -> ø

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

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 Sw21 Inh2 I2

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

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 Sw21 Inh2 DNA sense DNA temp BH-A

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

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

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

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