1. Harvard MIT DOE GtL Center Collaborating PIs: Chisholm, Polz, Church, Kolter, Ausubel, Lory arep.med.harvard.edu C.Ting 7-Feb-2005 4:10-4:40 PM 2-20 μm 0.6 μm

2. Molecular Systems Biology Access is free of charge Transcriptomics Proteomics Metabolomics Functional genomics Structural genomics Computational biology Theoretical biology Mathematical biology Synthetic biology www.nature.com/msb/

3. Harvard MIT DOE Center Projects ProchlorococcusPhotosynthesis, circadian & cell cycles EscherichiaSynthetic genomes/proteomes Vibrio4X faster replication than E.coli CaulobacterAsymmetric cell & chromsome structure PseudomonasBiofilms Poster# Topic Goal# 2. Leptos, et al. Proteomics1 121. Nguyen, et al. Mass spectrometry XML1 122. Nguyen, et al. Gene Regulation2 67. Thompson, et al. Vibrio diversity3 68. Martiny, et al. Prochlorococcus diversity3 77. Sullivan, et al. Cyanophage diversity1,3 3. Zhang, et al. Single cell sequencing1-4 1. Church, et al. Metabolic fluxes4 arep.med.harvard.edu

4. Prochlorococcus 40ºN - 40ºS Ocean chl a (Aug 1997 –Sept 2000) Provided by the SeaWiFS Project, NASA

5. Humans consume 2kW per person = 10 10 kW. Sunlight hits the earth at 40,000 times that rate (70% ocean). CO 2 370 ppm = 730 x10 15 g globally, increase ~3 x10 15 /yr. Ocean productivity = ~100 x10 15 g CO 2 /yr … due to Autotrophs: 10 26 Prochlorococcus cells globally (10 8 per liter) Sequestration v. respiration v. use: heterotrophs (Pelagibacter), phages, predators (Maxillopoda, Malacostraca, herring) Energy & CO 2 Sequestration http://www.gsfc.nasa.gov/gsfc/service/gallery/fact_sheets/earthsci/terra/earths_energy_balance.htm http://clear.eawag.ch/models/optionenE.html http://en.wikipedia.org/wiki/Copepod Morris et al. Nature 2002 Dec 19-26;420(6917):806-10. http://hosting.uaa.alaska.edu/mhines/biol468/pages/carbon.html http://www.aeiveos.com/~bradbury/Papers/PhotosyntheticEfficiency.html 0.1  0.1 mm 6 cm

6. Diel (circadian) cycle Light output for sun-box: 14hr light – 10hr dark, 230  E at peak Zinser, Lindell,Chisholm, Zinser, Lindell, Chisholm, Leptos, Jaffe, Lin,et al. Leptos, Jaffe, Lin, et al.

7. Light Dark Light Normalized expression Time (Hours) Diel Expression: All genes Zinser et al. unpubl.

8.  -Glc-1P ADP-Glc  -1,4-glucosyl-glucan glycogen Central Carbon Metabol. glgC glgX glgA glgB glgP Zinser et al. unpubl. Light regulated Prochlorococcus metabolism

9. Oxygenic Photosynthesis psbA =D1 D2 HLIP=HighLight Induced Protein Pc= Plastocyanin Fd=Ferridoxin H 2 OO 2 NADPH e - e - PSIPSII H 2 OO 2 H 2 OO 2 NADPH e - e - PSIPSII Core reaction Center Proteins

10. Photosynthetic Genes in Phage Podovirus P-SSP7 46 kb PCHLIPsFdD1 12kb 24kb PCHLIPsFdD1 12kb 24kb ~500bp HLIPsD1D2 6.4kb2.8kb ~500bp Myovirus P-SSM4 181 kb HLIPsD1D2 6.4kb2.8kb Lindell, Sullivan, Chisholm et al. 2004 HLIPD1 Myovirus P-SSM2 255 kb

11. RNA Responses to Phage MED4-0682 (60 aa Conserved URF) Phage SSP7 psbA MED4 host psbA Lindell,Sullivan, Zinser, Chisholm Lindell, Sullivan, Zinser, Chisholm

12. Synthetic - homologous recombination testing of DNA motifs 1.3 2.4 (1.3 in  argR) 1.1 1.3 0.7 2.5 0.2 1.4 1.4 3.5 RNA Ratio (motif- to wild type) for each flanking gene Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208

13. Synthetic Genomes & Proteomes. Why? Test or engineer cis-DNA/RNA-elements Access to any protein (complex) including post-transcriptional modifications Affinity agents for the above. Protein design, vaccines, solubility screens Utility of molecular biology DNA -- RNA -- Protein in vitro "kits" (e.g. PCR -- T7 -- Roche) Toward these goals design a chassis: 115 kbp genome. 150 genes. Nearly all 3D structures known. Comprehensive functional data.

14. (PURE) translation utility Removing tRNA-synthetases, translational release-factors, RNases & proteases Allows: Selection of scFvs[antibodies] specific for HBV DNA polymerase using ribosome display. Lee et al. 2004 J Immunol Methods. 284:147 Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353 High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al. 2004 Eur J Biochem 271:568 Cell-free translation reconstituted with purified components. Shimizu et al. 2001 Nat Biotechnol. 19:751-5. Also: membrane incompatible expression & diverse amino-acids (>21)

15. in vitro genetic codes 5' mS yU eU UGG UUG CAG AAC... GUU A 3' GAAACCAUG fMTNVE | | | 5' Second base 3' U A C C U mS yU eU A C U G A Forster, et al. (2003) PNAS 100:6353 Zhang et al. (2004) Science. 303:371 80% average yield per unnatural coupling. eU = 2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid mS = O-methylserine gS = O-GlcNAc–serine bK = biotinyl-lysine

16. Forster & Church Oligos for 150 & 776 synthetic genes (for E.coli minigenome & M.mobile whole genome respectively)

17. Up to 760K Oligos/Chip 18 Mbp for $700 raw (6-18K genes) <1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (U.Houston) 24K Agilent Ink-jet standard reagents 48K Febit 100K Metrigen 380K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church

18. Improve DNA Synthesis Cost Synthesis on chips in pools is 5000X less expensive per oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!) Solution: Amplify the oligos then release them. 10 50 10 => ss-70-mer (chip) 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng, Zhou, Gulari, Gao, Church Nature 2004 => ds-90-mer => ds-50-mer

19. Improve DNA Synthesis Accuracy via mismatch selection Tian & Church Other mismatch methods: MutS (&H,L)

20. Computer Aided Design Polymerase Assembly Multiplexing (CAD-PAM) Moving forward: 1. Tandem, inverted and dispersed repeats (hierarchical assembly, size-selection and/or scaffolding) 2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates 3. 15kb to 5Mb by homologous recombination (Nick Reppas) 4. Phage integrase site-specific recombination, also for counters. Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB. 50 75 125 225 425 825 … 100*2^(n-1)

21. All 30S-Ribosomal-protein DNAs (codon re-optimized) Tian, Gong, Sheng, Zhou, Gulari, Gao, Church 1.7 kb 0.3 kb s19 0.3kb Nimblegen 95K chip Atactic <4K chip

22. Improving synthesis accuracy Method Bp/error Chip assembly (PAM) 160 1 Hybridization-selection 1,400 1 MutS-gel-shift 10,000 2 MutHLS cleavage 30,000 3 (10X better than PCR) 1. Tian, Church, et al. 2004 Nature 432:1050 2. Carr, Jacobson, et al. 2004 NAR 32:e162 3. Smith & Modrich 1997 PNAS 94:6847

23. Extreme mRNA makeover for protein expression in vitro RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable. Solution: Iteratively resynthesize all mRNAs with less mRNA structure. Tian & Church Western blot based on His-tags

24. Safe Synthetic Biology Church, G.M. (2004) A synthetic biohazard non-proliferation proposal. http://arep.med.harvard.edu /SBP/ Church_Biohazard04c.dochttp://arep.med.harvard.edu /SBP/ Church_Biohazard04c.doc 1. Monitor oligo synthesis via expansion of Controlled substances, Select Agents, &/or Recombinant DNA 2. Computational tools are available; very small number of reagent, instrument & synthetic gene suppliers at present. 3. System modeling checks for synthetic biology projects 4. Multi-auxotroph, novel genetic code for the host genome, prevents functional transfer of DNA to other cells.

25. Marine Synechococcus high light adapted Prochlorococcus low light adapted Prochlorococcus MIT9201 GP2 MIT9107 SAR6 TATL1a ENATL1 ENATL3 MIT9302 MIT9312 MIT9202 MIT9215 TATL1b Pac 1 ENATL7 ENATL4 MIT9211 MIT9303 SAR139 WH8112 SAR100 WH8101 WH8012 WH7805 SAR7 Synechococcus PCC6307 0.01 89 97 92 72 71 100 78 66 84 Photosynthetic bacterial genomes (for population genetics & proteomics) MED4 NATL2A SS120 MIT9313 WH8102

26. Monthly samples Isolate Vibrios Identity population as cluster of barcode genes Quantification: population is continuously present Genomes: almost each genome different in typical sample Additional marker gene: highly diverse Environmental population genomics (of a ribotype cluster) Thompson, Polz, et al. (2005) Science

27. Sequencing single cells Biome studies focus on single-cells because hard to grow in the lab, multiple DNAs & RNAs per cell, exchange genome subsets. (Complementary to Biome shotgun and/or 100 kb BACs) Many input molecules required to sequence one molecule. vs. one molecule sufficient to sequence via many copies of it.

28. Amplifying DNA from single cells  29 real-time amplification No template control Affymetrix quantitation of independent amplifications Prochlorococcus & Escherchia Zhang, Martiny, Chisholm, Church, unpub.

29. Polony Bead Sequencing Pipeline In vitro libraries via paired tag manipulation Bead polonies via emulsion PCR [Dre03] Monolayered immobilization in acrylamide Enrichment of amplified beads SOFTWARE Images → Tag Sequences Tag Sequences → Genome FISSEQ or “wobble” sequencing Epifluorescence Scope with Integrated Flow Cell Mitra, Shendure, Porreca, Rosenbaum, Church unpub.

30. Read length needs for population surveys Paired tags are separated by 1000 +/- 100 bases

31. Polony Fluorescent In Situ Sequencing Libraries Greg Porreca Abraham Rosenbaum 1 to 100kb Genomic M L R M PCR bead Sequencing primers Selector bead 2x20bp after MmeI ( BceAI, AcuI) Dressman et al PNAS 2003 emulsion

32. Cleavable dNTP-Fluorophore (& terminators) Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65 Reduce or photo- cleave

33. Polony- FISSeq : up to 2 billion beads/slide Cy5 primer (570nm) ; Cy3 dNTP (666nm) Jay Shendure Self Organizing Monolayer

34. High accuracy special case: homopolymers (e.g. AAA, CC, etc.) Use "compressed" tags, ACG = ACCG=ACCCG Quantitate incorporation Reversible terminators FRET between adjacent 3' bases Wobble primers, CTAGCGAGCTAGNNNNNNNNA All five of these work. Maintenance of amplification fidelity using linear amplification from initial genomic fragment

35. # of bases sequenced (total)23,703,953 # bases sequenced (unique)73 Avg fold coverage324,711 X Pixels used per bead (analysis)~3.6 Read Length per primer14-15 bp Insertions 0.5% Deletions 0.7% Substitutions (raw) 4e-5 Throughput:360,000 bp/min Polony FISSeq Stats Current capillary sequencing 1400 bp/min (600X speed/cost ratio, ~$5K/1X) (This may omit: PCR, homopolymer, context errors) Shendure

36. Wobble vs Simple primer sequencing 1 vs 2.5 bp read/cycle of 4 bases 10 vs 14-200 bp reads 3e-3 vs 4e-5 non-homopolymer errors 3e-3 vs 1e-1 homopolymer errors 40 minutes per base tested = 60 hr per 20 cycles (20 hr, if 4 colors)

37. Harvard MIT DOE Center Projects ProchlorococcusPhotosynthesis, circadian & cell cycles EscherichiaSynthetic genomes/proteomes Vibrio4X faster replication than E.coli CaulobacterAsymmetric cell & chromsome structure PseudomonasBiofilms Poster# Topic Goal# 1. Church, et al. Metabolic fluxes4 2. Leptos, et al. Proteomics1 68. Martiny, et al. Prochlorococcus diversity3 121. Nguyen, et al. Mass spectrometry XML1 122. Nguyen, et al. Gene Regulation2 77. Sullivan, et al. Cyanophages1,3 67. Thompson, et al. Vibrio diversity3 3. Zhang, et al. Single cell sequencing1-4 arep.med.harvard.edu