Synthesis of useful replicating biosystems

1. Synthesis of useful replicating biosystems 28-Jan-2004 DOE Thanks to: DOE-HGP 1987-2002 DARPA 2001-3 (* Currently unfunded* )

2. DOE Synthetic Genomes: Why? Cheaper/faster "standard biology", hypothesis testing Systems Biology: Multiple simultaneous tests Viruses: Aid strain transfer; generate variants, new haplotypes Anti-viral vaccines and therapeutics (including variants) In vitro: Make products toxic in E.coli. Microbes: Interspecific hybrids (e.g. codon usage) Structural biology: variants Rapid vaccine response to engineered bioterrorism. Cell-mediated immunity + humoral. Fix mismatch between genome analysis & synthesis

3. DOE Synthetic Genomes: Why? In vitro Microbial & Human Antimutators Artificial ecosystems (laboratory scales) Energy aiding pathway improvement Instrustrial production: Enzymes, SingleCellProtein, Protein-drugs Remediation: Hybrid genomes (opt. codons), combinatorial pathway (Maxygen & Diversa). Xylose & Oil Pharmaceuticals: Combinatorial syntheses Nano science Combinatorial syntheses, Complex nanosystems, more general nanoassembly (in reach of polymerases and ribosome-like factories) Health research: 10X faster results per current $ (cost/benefit) Hypothesize & test unknown gene combinations Synthetic standards (arrays, MS, quantitation, etc) Agriculture: salt, cold, drought, pest tolerant hybrid genomes

4. DOE Synthetic Genomes I. Charge & Intro. David & Ray A. Follow on to genome project B. DOE role in tech development in Biology & HGP C. Thumbnail of DOE missions: bioremediation, energy, carbon sequestration II. State of the Art of Technology George III. New DOE technology. George A. What is new? B. Near and far future. 1&10 yr. IV. Benefits & Concerns 1&10 yr. A. GMO-gene escape (old tech) Jim B. Bioterrorism C. Ownership D. Patents/IP

5. DOE Synthetic Genomes IV. Benefits & Concerns: Generic, DOE, other A. GMO-gene escape (old tech) Eliminate allergenic (interspecies) pollen B. 10X greater yield for constant research $ (hypothesis testing & systems biology) C. Pharma throughput D. Vaccines variants F. Bioterrorism (access/regulation, biohacker/vandals) DEA model for tracking chemicals tracking bioprinter cell design output (is this to hard?) G. Ownership, Patents/IP, Research sociology, agenda H. spin-offs: e.g. I/O memory nm3 vs. micron3 I. Religion life (refer to Cho et al. 1999)

6. Energy & CO2 Fluxes 4x1013 kW of sunlight hits earth. We consume 2kW per person* 6x109 = 1010 kW. CO2 >370 ppm = 730 x1015 g globally, increase ~3 x1015 /yr. Ocean productivity = ~100 x1015 g/yr. Autotrophs: 1025 Prochlorococcus cells globally (108 per liter) Undone by Cyanophages & Heterotrophs: 2x1028 SAR11 cells in the oceans Pseudomonas & Caulobacter in a variety of soils & aquatic environments http://www.gsfc.nasa.gov/gsfc/service/gallery/fact_sheets/earthsci/terra/earths_energy_balance.htm http://clear.eawag.ch/models/optionenE.html Morris et al. Nature 2002 Dec 19-26;420(6917):806-10. http://hosting.uaa.alaska.edu/mhines/biol468/pages/carbon.html

7. Synthetic Genomes: How? (examples) How to decrease cost? Chips, error correction, polony clone/sequence How to make new biopolymers? Altered translation, mirror proteins How to improve energy production/conservation? Eliminate predation How to increase safety? Eliminate DNA/RNA exchange In vivo vs in vitro? ribosome-display selection, in vivo assembly

8. Transition L-amino acids & D-ribose (rNTPs, dNTPs) Transition EF-Tu, peptidyl transferase D-amino acids & L-ribose (rNTPs, dNTPs)

9. Impact of mirror cells? Microbes Eliminate DNA exchange Energy Prochlorococcus resistant to phage & predators Remediation Engineer community resistant to predation Pharmaceuticals Expand "natural products" Nano science Enzyme resistant "bio"-polymers Health Mirror humans resistant to all? viruses ? Viruses see above Agriculture Pest resistance

10. Synthetic Biology • Test or manipulate optimality • Program minimal cells (105kbp) • Nanobiotechnology - new polymers • Manage complex systems • e.g. stem cells & ocean ecology

11. Synthetic Genomes • Molecular Biology depends on • in vitro reactions (e.g. PCR, SP6, Roche) • Utility of mirror-image & other unnatural • polymers. • Combine with homologous recombination to engineer larger genomes • Toward these goals design a minimal chassis: • 100kbp genome. • All 3D structures known. • Comprehensive functional data.

12. Known: in vitro assembly & 3D structure of prokaryotic ribosomes (e.g. Nomura et al.; Noller et al.)

13. A 105 kbp mini-genome

14. The least well-characterized components of the mini-genome Forester & Church

15. All 30S-Ribosomal-protein DNAs & mRNAs synthesized in vitro M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 DNA Template RNA Transcript Tian & Church

16. His-tagged ribosomal proteins synthesized in vitro RS-2,4,5,6,9,10,12,13,15,16,17,and 21 as original constructs. RS1 required deletion of a feedback motif in the mRNA. RS-3, 7, 8, 11, 14, 18, 19, 20 are still weakly expressed. Note that S1, S4, S7, S8, S20, L1, L4, L10 are known to repress their own translation (and are likely titrated by rRNA). Iteratively resynthesize all mRNAs (e.g. with less structure). Tian & Church

17. Custom Oligonucleotide Chips *Photo-Generated Acid Electrolytic acid/base Photolabile 5'protecting group Ink-jet with standard monomers and acids *http://www.xeotron.com/fw/main/default.asp http://www.oxamer.com/ http://www.febit.com/geniom/go_DNAProc.htm http://www.nimblegen.com/ http://www.chem.agilent.com/Scripts/PDS.asp?lPage=3071 Tian, Sheng, Gao & Church

18. Improving synthesis fidelity

19. Why mismatch repair works for in vivo replication (& not for de novo synthesis) Desired (or original) base pair: AT ; Mutant bp: gc Heteroduplexes: Ac & gT. Mismatch repair (selecting original): At & aT Mismatch repair (no "original"): At, aT, gc, gc Solution: Select on majority (once or more) (1) Kinetic: + (AT) -(gc, AC , gT) (2) Equilibrium: + (AT , gc) -(Ac , gT)

20. Improving automated cost