Past & Near-Future Synthetic Biology Projects

Past & Near-Future Synthetic Biology Projects iGEM Harvard Thu 2-Jun-2005 10:00-10:30 AM Thanks to:Washington U, Harvard-MIT Broad Inst.,DARPA-BioSpice, DOE-GTL,EU-MolTools, NGHRI-CEGS, NHLBI-PGA, NIGMS-SysBio,PhRMA, Lipper Foundation Agencourt, Ambergen, Atactic, BeyondGenomics, Caliper, Genomatica, Genovoxx, Helicos, MJR, NEN, Nimblegen,SynBioCorp, ThermoFinnigan, Xeotron/Invitrogen For more info see: arep.med.harvard.edu

Avoiding the tarpits How experiments can go wrong: 1. Abstraction: decouple design/fabrication 2. Protein overproduction 3. Protein destabilizers How they go right: 1. Repeating previous work 2. Keep it complex 3. Training pets to do what they do naturally Reliable methods under-utilized in iGEM: 1. PCR 2. Recombineering 3. Selection

Integration & details Basic lab hygiene: 1. Positive AND negative controls 2. Communicate quantitatively (including variance) 3. Quality & testing from start (& continuity) Incompatibilities: 1. Intra/extra/non-cellular (e.g. redox, detergents) 2. Moving a portion of an interacting set (e.g. promoters) 3. Codon usage 4. Protein stability, aggregation 5. Cross-talk (always plan "background")

Engineering Biological Systems Action Specificity %KO "Design" Small molecules (drugs)Fast Varies Varies Hard Antibodies Fast Varies Varies Hard RNAi Slow Varies Medium OK Riboregulators Fast Varies Medium + /- Insertion "traps" Slow Yes Varies Random Recombination Slow Perfect Complete Easy Proteasome targeting Fast Excellent Medium Easy Physical environment Varies Microfabrication Varies

Proteasome targeting(via drug + homologous recombination) Janse, DM, Crosas,B Finley,D & Church, GM (2004) Localization to the Proteasome is Sufficient for Degradation.

Programmable ligand-controlled riboregulators of eukaryotic gene expression. Bayer & Smolke 2005Nat Biotech. 23:337-43.

Integrase Counter Team (IGEM Summer '04) http://theory.med.harvard.edu/SynBio/ Harvard University • John Aach • Patrik D'haeseleer • Gary Gao • Jinkuk Kim • Xiaoxia Lin • Nathan Walsh • George Church Boston University • Will Blake • Jim Flanigon • Farren Isaacs • Ellen O’Shaughnessy • Neil Patel • Margot Schomp • Jim Collins Gardner et al.2000 Nature 403:339 Construction of a genetic toggle switch in E.coli

Int Xis TF4 1 Int Xis TF3 2 Int Xis TF5 3 Int Xis TF6 4 In vivo Counter Designs 1 2 3 • Riboswitch • counter  • 0 • 1 • 1 • 0 • Integrase bit counter • Cell-cycle counter 

Integrase advantages • High fidelity – site specific and directional recombination (as opposed to homologous recombination) • Reversible – excision just as reliable as integration • Specific – each integrase recognize its own att sites, but no others • Numerous – over 300 known Tyr integrases and ~30 known Ser integrases • Efficient – very few other factors needed to integrate or excise • Extensively used – Phage systems well characterized and used extensively in genetic engineering (e.g., the GATEWAY cloning system by Invitrogen)

Integrase/Excisionase structure • l Integrase (int) • l Excisionase (xis) • Mol Cell. 2003 Jul;12(1):187-98. A conformational switch controls the DNA cleavage activity of lambda integrase. Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T. • Sam MD, Cascio D, Johnson RC, Clubb RT. Crystal structure of the excisionase-DNA complex from bacteriophage lambda. J Mol Biol. 2004 Apr 23;338(2):229-40.

Design Phage Int/Xis system Phage attachment sites attP O P P’ O B B’ attB Bacterial attachment sites Int + Xis Int Integrated Left attachment sites attL Integrated Right attachment sites attR O O B P’ P B’ Stably integrated prophage

Int/Xis system with inverted att sites Phage attachment sites attP Bacterial attachment sites attB* 0 O O P P’ B B’ Int + Xis Int Integrated Right attachment site attR Integrated Left attachment site attL* 1 O O P P’ B B’

Design 1: Bit counter initial concept 0 0 0 0 • Counting mechanism: • Initial state: 0 0 0 • Pulse 1: 1 0 0 • Pulse 2: 0 1 0 • etc. . . . • Race condition problems between each Int and Xis 1 1 Int1 Xis1 Int2 Xis3 Int2 Xis2

Int2 Int2 Xis2 Rpt1 int2 xis2 rpt1 int1 xis1 reporter2 attR2 – – attL2* term int2 xis2 reporter1 attR1–term– attL1* Int1 Xis1 Rpt2 Int1 int1 xis1 rpt2 int1 int1 xis1 reporter2 int2 xis2 reporter1 attP2–term–attB2* attP1– – attB1* term Design 2: Full Cycle of Two ½-bits int2 int2 xis2 reporter1 1 attR1–term– attL1* int1 xis1 reporter2 2 attP2–term– attB2*

BioBricks

Design Composite half bits in BioBricks Two 2kb composite parts: λ Int+ LVA p22 attP Reverse Terminator p22 attB (rev comp) λXis +AAV ECFP +AAV λHalf Bit BBa_I11060 : BBa_I11020 BBa_I11033 BBa_B0025 BBa_I11032 BBa_I11021 BBa_E0024 p22 Int+ LVA λattP Terminator λattB (rev comp) P22 Xis +AAV EYFP +AAV p22 Half Bit BBa_I11061 : BBa_I11030 BBa_I11023 BBa_B0013 BBa_I11022 BBa_I11031 BBa_E0034 • Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29, 2205-2216 • Andersen, Applied and Environmental Microbiology, 1998, 2240-2246

Integrase counter: ODE & Stochastic modeling The simulation is sensitive to the relative degradation rates of Int and Xis. Previously Int was less stable, but in this simulation the stabilities are equal.

Synthesis & Testing:Can Int + Xis control GFP expression? PLlacO PLtetO attP Int GFP_AAV attB* pBAD Xis pSC101 Kan • Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25, No. 6 1203-1210

Invitrogen Gateway Vectors Parr RD, Ball JM.(2003) Plasmid 49:179. Nakayama M, Ohara O. (2003) BBRC 312:825

SsrA 11-aa 'lite' tags reduce repressor half-life from > 60 min to ~4 min. A synthetic oscillatory network of transcriptional regulators Insets: normalized autocorrelation of the first repressor Continuous model Stochastic similar parameters Elowitz &Leibler, (Pub), Nature 2000;403:335-8

Synthetic oscillator network Controls with IPTG Variable amplitude & period in sib cells Single cell GFP levels Elowitz &Leibler, Nature 2000;403:335-8

Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in vitro Nakajima, et al. Science. 2005 Apr 15;308(5720):414-5

Past & Near-Future Synthetic Biology Projects