1. 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 iGEM Harvard Thu 2-Jun-2005 10:00-10:30 AM Past & Near-Future Synthetic Biology Projects

2. 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

3. 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")

4. 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

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

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

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

8. In vivo Counter Designs XisTF4 XisTF3 Int XisTF5 XisTF6 Int 1 2 3 4 Riboswitch counter   Integrase bit counter Cell-cycle counter  01100110 123123

9. 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)

10. Integrase/Excisionase structure 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. Integrase (int) Excisionase (xis)

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

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

13. Design 1: Bit counter initial concept 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 Int 1 0 0 0 Xis 1 Int 2 Xis 2 Int 2 Xis 3 11 0

14. Design 2: Full Cycle of Two ½-bits StatePulseProducts 0 0 1AInt 2 0 1 2A Int 1 Xis 1 Rpt 2 1 1 1B Int 2 Xis 2 Rpt 1 1 0 2BInt 1 0 0 1 xis 2 reporter 1 int 2 2 xis 1 reporter 2 int 1 attR 1 –term– attL 1 * attP 2 –term– attB 2 * int 2 Int 2 int 1 Int 1 xis 1 Xis 1 rpt 2 Rpt 2 int 2 Int 2 xis 2 Xis 2 rpt 1 Rpt 1 int 1 Int 1 xis 1 reporter 2 int 1 attR 2 – – attL 2 * term xis 1 reporter 2 int 1 attP 2 –term– attB 2 * attP 1 – – attB 1 * xis 2 reporter 1 int 2 term xis 2 reporter 1 int 2 attR 1 –term– attL 1 *

15. BioBricks Basic Part TypesComposite Part Types TypeDescriptionTypeDescription RRegulatoryOperator regionEReporterCompound reporter devices BRBSRibosome binding siteQInverterInverter and logic CCDSProtein coding sequence CompositeOther composite parts BTerminatorTranscriptional terminatorIProjectStudent projects RNA RNA binding sites and coding GGeneratorTIPS-to-Protein converter FSignallingCell-cell signalling Measurement Performance measurement constructs EReporterBasic reporter CDSTTemporaryTemporary and trial parts MTagTag or ModifierSIntermediateGenerated during assembly VPlasmidPlasmids OtherParts not yet classified VCellsCell strains

16. Design Composite half bits in BioBricks λ Xis +AAV ECFP +AAV λ Int+ LVA BBa_E0024BBa_I11020BBa_I11021 p22 attP BBa_I11033 Reverse Terminator BBa_B0025 p22 attB (rev comp) BBa_I1103BBa_I11032BBa_I11060BBa_I11060 : P22 Xis +AAV EYFP +AAV p22 Int+ LVA BBa_E0034BBa_I11030BBa_I11031 λ attP BBa_I11023 Terminator BBa_B0013 λ attB (rev comp) BBa_I11022BBa_I11061BBa_I11061 : Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29, 2205-2216 Andersen, Applied and Environmental Microbiology, 1998, 2240-2246 Two 2kb composite parts: λ Half Bit p22 Half Bit

17. 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.

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

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

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

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

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

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