Constructionism S parts NRB 258 Thu 11 -Jul-2005

Constructionism: S parts NRB 258 Thu 11 -Jul-2005 12: 30 -2 ish PM Collaborators Grants Tech Transfer Courses Past Present Future Biological Analytic Synthetic Computational Citizenship

Why discuss the big picture? What do I do? What do we do? Grants & reviews. Recruit Faculty, postdocs, students, staff TAC, PQE, courses, symposia, press Lab, dept. , school, international policy Editor time & quality Companies, IP, secrecy, competition So why am I still smiling? http: //www. chez. com/cropcircles/Nazca. htm Invention. Sharing. Teamwork. Integration as a specialty. Grand Challenges. Quantitative not qualitative. Quality/$ not quantity/sec. Automation is economics, but also epistemology of our craft.

Grand Challenges • Infection-proofing Vaccines New genetic codes , chirality • $1 K Genome Bioweathermap • Low-senescence, low-cancer stem cells • Neuroimaging • Autofabrication • Space

3 Exponential technologies (synergistic) Computation & Communication (bits/sec~m$) urea Synthesis (amu/project~M$) E. coli operons t. RNA B 12 telegraph Analysis (kamu~base/$) t. RNA Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

Present http: //arep. med. harvard. edu/gclab 3. htm (MS: Kyriacos Leptos, Michael Chou, Dat Nguyen) (Syn. Reg: Aimee Dudley, Priya Dutta, Noel Goddard, Dana Pe'er, Daniel Janse) (Metab: Aaron Brandes, Xiaoxia Lin, Gary Gao, Wayne Rindone, Jeremy Zucker) (Plone: Greg Porreca, Abraham Rosenbaum, Jay Shendure, Kun Zhang, Shawn Douglas, Joseph Chou, Michael Wang, Madeleine Price, Chris Varma) (Adnan Derti, Pedro de Magalhaes, Madhu Nikku, Dheeresh Patel) (Syn. Bio: Farren Isaacs, Nick Reppas, Jingdong Tian, Sasha Wait, Hui Gong, John Tsang) (Cindy Vallaro, Mayra Mollinedo) (Imaging: John Aach, Natasha Novikov, Benjamin Williams) (Motifs: Rhonda Harrison, Allegra Petti, Zhou Zhu, Julie Greenberg) (3 DReg: Mark Umbarger, Matthew Wright, Peter Kharchenko) Summer'05: Nomso Agunwamba, Resmi Charalel, Yetunde Ibrahim, Michelle Kuykendal, Mirko Palla, Xavier Rios New Fellows: Andy Levin, Gil Alterovitz, Jayant Rajan

Shared interests http: //arep. med. harvard. edu/gmc/collab. html Daley Stem/germ-cell DNA methylation, cancer drug resistance Struhl Human haematopoetic stem cell model Mitra, Gottlieb, Sherley Polonies & Stem Cells Chisholm, Polz, JGI Ruvkun Ausubel, Kolter, Lory Laub, Dekker Perrimon Wu Letvin Jacobson Single-cell sequencing Prochlorococcus Environmental microbes & Mars Biofilms 3 D chromosome RNAi & Imaging Pairing & homologous recombination T-cell recognition of HIV gag x MHC diversity r. E. coli (re-engineered E. coli)

Technology Transfer http: //arep. med. harvard. edu/gmc/tech. html CRI '61 GTC Agencourt Collab. Diag Oscient. PC '93 '98 GPC '03 APG Beckmann-Coulter '05 Seq: Ambergen, Helicos, Caliper, MJR, NEN, Agencourt. Syste. MS: Beyond. Genomics, Thermo. Finnigan, Genomatica Syn: Xeotron/Invitrogen/Atactic, Nimblegen, Codon. Devices SAB: David Baker(UW), George Church(HMS), Jim Collins(BU), Drew Endy(MIT), Mike Elowitz(Caltech), Mike Hunkapiller (Alloy), Joe Jacobson(MIT), David Liu(Harvard), Jay Keasling(UCB), Paul Modrich(Duke), Christina Smolke(Caltech), Ron Weiss(Princeton).

Biological bases Prochlorococcus Caulobacter Escherichia Saccharomyces Mus Homo 1. 7 M 4. 0 M 4. 8 M 12. 0 M 3. 0 G Solar energy & CO 2 fix 3 D chromosome & cell structure Genetic codes Regulatory codes Embryonic stem cells Cancer & personal genomics

Technological 73 -74 Mycoplasma 74 -77 Computational crystallography: CORELS, 1 st folded-NA 77 -78 DNA lattices & sequencing: 1 st plasmid 78 -80 RNA&DNAsplicing: ribozymes & meganucleases 80 -85 Genomic sequencing: Ig-enhancers, 5 m. C 84 -86 Embryonic stem cells 86 -94 Multiplexing: 1 st genome Helicobacter 89 -05 Proteomics (E. coli, Mycoplasma, stem cells) 91 -05 Oligo array synthesis 95 -05 RNA arrays 97 -05 Polonies chromosome fold/pair 01 -05 Synthetic Biology (&lattices) 04 -05 Stem cells epigenetics, 04 -05 Personal (open) Genomics

Computational 74 -77 3 D DNA/RNA/protein modeling 77 -86 Seq-imaging, motifs, DNA-design-editors 86 -95 Automation & GUIs 95 -98 Gene finding, proteomics 97 -05 Clustering, DNA motifs 01 -05 FBA, ODE, Sys. Bio 03 -05 Syn. Bio design CAD 03 -05 Personal Genomics & imaging

Grants http: //arep. med. harvard. edu/gmc/sponsor. html 86 -97 HHMI (partial list) 87 -02 DOE-HGP (90 - GTC, MIT, Stanford HGP) 97 -01 Lipper Foundation 98 -01 Aventis 98 -01 DARPA-Ultrascale computing 01 -05 DARPA-Bio. Spice 01 -04 NHLBI-PGA (Seed/Seigo/Seidman) 02 -07 DOE-GTL (04 Syn. Bio supplement) 02 -05 Ph. RMA 02 -05 Stem cells (Zon) Computional 03 -08 NIGMS-Sys. Bio (Murray) 04 -09 NGHRI-CEGS Polony/Stem cells 06 -11 NHGRI (Jacobson) 06 -11 NSF (Keasling) 06 -11 NHGRI (Forster) 06 -08 Harvard Stem Cell Inst. & Bio. Engineering

Synthetic Biology Synth chem & biomimetics Genetic engineering Analytic chem & omics Systems biology & protein design Synthetic Genomics Synthetic regulatory circuits Sensors Energy Metabolic engineering Minimal genomes Drug biosynth Synthetic proteomics Materials Vaccines

Custom chip projects Nimblegen http: //arep. med. harvard. edu/web 2/chips. htm analytic Xeotron Combimatrix &Quality &Selection Design Ordered Size When File Received Project 2003? Lindell 200 K? 2003? Na Y Prochlorococcus analytic 1 -Feb-04 Tian 918 1 -Feb-04 XQS Y Ribosomal proteins 25 -Mar-04 Tian 95, 376 25 -Mar-04 NS Y Ribosomal proteins 28 -Feb-05 Isaacs 117, 000 4 -Mar-05 NQS Y r. E. coli genome 6 -May-05 Bradbury 15, 579 6 -May-05 NQS ? ? Coral fluorescent protein combos 6 -May-05 Tian ? 6 -May-05 N Y CD 44 exon combos 19 -Jun-05 Mitra 342, 835 19 -Jun-05 N- - Human exon primers 24 -May-05 Norville 1, 027 30 -Jun-05 C- - S-layer crystals ND Price ? ND Na - DNA methylation analytic 9 -May-05 Reppas 364 30 -Jun-05 C- - Pos/neg-selection system 4 -May-05 Sasha 71, 727 30 -Jun-05 N - Bit-counter ND Aimee 2000? ND C - Yeast promoters ND Lin ? ND ? - Multi-phospho-protein kinetics

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

DNA motif target & quantitative effects (via homologous recombination) 2. 4 (1. 3 in Darg. R) 1. 3 1. 1 1. 3 2. 5 0. 7 0. 2 1. 4 3. 5 RNA Ratio (motif- to wild type) for each flanking gene Bulyk, Mc. Guire, Masuda, Church 2004 Genome Res. 14: 201– 208

Why Synthetic Genomes & Proteomes? • Test or engineer cis-DNA/RNA-elements • Drug biosynthesis e. g. Artemesinin (malaria) • Epitopes & vaccines. • Unnatural aa & post-translational modifications • De novo protein design & selection. • Humanizing imm/tox systems, E. colizing codons • 20 bit in vivo counters • Why whole genomes? Changing the genetic code, safety, genome stability, enhanced restriction, recombination

Constructing new genetic codes (two examples) 1. Codons: 313 UAG stop > UAA stop 2. Delete RF 1 (1 free codon, for new aa e. g. PEG-p. Ac. Phe-h. GH) 1. 2. 3. 4. 5. Codons: AGY Ser > UCX Ser t. RNAs: AGY Ser > AGY Leu Codons: UUR/CUX Leu > AGY Ser t. RNAs: UUR Leu > UUR Ser Codons: UCX Ser > UUR Ser (Leu & Ser now switched & 8 codons free)

Mirror world : enzyme, parasite, & predator resistance & access 2 n diastereomers (n chiral atoms) 1. Transition mutants: EF-Tu, peptidyl transferase, pol/ligase with D-amino acids & L-r/d. NTPs: Dedkova et al. Enhanced D-amino acid incorporation into protein by modified ribosomes. JACS 2003 125, 6616. Semizarov et al. Stereoisomers of d. NTPs as substrates. . J Biol Chem. 1997 272: 9556. 2. De novo chemistry: Milton et al. Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity 1992 Science 256: 1445; Urata et al. Synthesis and properties of mirrorimage DNA. 1992 NAR 20: 3325.

Mirror world : http: //www. chemgenes. com/ANP-8034. php Thymidine cyanoethyl di-isopropyl phosphoramidite b-L (synthetic) b-D (natural)

5 Mbp Genome assembly alternatives 1. cat 2. kan 3. Automated in vivo homologous recombination: Serial electroporation: 48 stages: 1 strain (21 hr/stage) vs. Hierarchical conjugation: 7 stages: 48 > 24 > 12 > 6 > 3 > 2 > 1 strains vs. Random/simultaneous 1 or more stages Reppas & Church cat

Hierarchical de novo DNA assembly Conditional Selectable genes: 2 = chloramphenicol, 3 = kanamycin (better: 2=sup. F, 3=ta. RNA) Conditional Meganuclease sites: 4 = I-Sce. I taggg_ataa^cagggtaat 5 = I-Dmo. I gccttgccgg_gtaa^gttccggcgcg Conditional Conjugative transfer elements: 6 = Col. E 1 ori. T 7 = F (inc. BCD) ori. T Condititional origins of replication (or in main chromosome ori. C) 8 = Inc. X ori-R 6 K (pir protein) 9 = Inc. Pα ori. V (trf. A protein).

Hierarchical de novo DNA assembly selection=2, 3 cut sites=4, 5 transfer=6, 7 replication=8, 9 84 ab-2 yz 46 95 -bc 3 yz 57 95 cd-3 yz 57 84 -de 2 yz 46 95 ef-3 yz 57 84 -fg 2 yz 46 84 gh-2 yz 46 95 -hi 3 yz 57 84 ij-2 yz 46 95 -jk 3 yz 57 95 kl-3 yz 57 84 -lm 2 yz 46 95 mn-2 yz 57 84 -no 3 yz 46 84 op-3 yz 46 95 -pq 2 yz 57 100 kb 84 abc 3 yz 46 95 cde 2 yz 57 84 abcde 2 yz 46 95 efg 2 yz 57 84 ghi 3 yz 46 95 efghi 3 yz 57 84 abcdefghi 3 yz 46 84 ijk 3 yz 46 95 klm 2 yz 57 84 ijklm 3 yz 46 95 mno 3 yz 57 84 opq 2 yz 46 95 mnopq 2 yz 57 95 ijklmnopq 2 yz 57 84 abcdefghijklmnopq 2 yz 46 200 kb 400 kb 800 kb 1. 6 Mbp

Sequence monitoring of evolution (anticipate escape & resistance)

Safer biology via synthetic biology difficulty • Systems modeling • Hi. Fi gene replacement • Inexpensive bio-weather-map custom biosensors (airborne & medical fluids), • International bio-supply-chain licensing (min research impact, max surveillance) • Metabolic dependencies prevent survival outside of controlled environments • Multi-epitope vaccines & biosynthetic drugs. • Cells resistant to most existing viruses via codon changes see: arep. med. harvard. edu/SBP

Responsible Conduct In Research "If scientists find that their discoveries have implications for some important aspect of public affairs, they have a responsibility to call attention to the public issues involved. . A good example is the response of biologists to the development of recombinant DNA technologies -- first calling for a temporary moratorium on the research and then helping to set up a regulatory mechanism to ensure its safety. " http: //www. aaes. org/membership/index. asp http: //www. nap. edu/readingroom/books/obas/

Education, journals, press 78 Bch 212 Molecular Biology (Wiley , Harrison alternate years) 88 -98 Gen 210 Conceptual Foundations of DNA Research 99 -03 Bph 101 Genomics & Computation Biology 04 -05 i. GEM: Genetically Engineered Machines 05 Bph 101 Genomics, Computing, Economics & Society BMC-Bioinformatics (impact factor - 5. 42) Omics Nature-EMBO-Molecular Systems Biology

Genome Analysis Policy • Insurance/employment: What probability & level of advantage can be hidden/examined? • Individual/group stigma • Choice, stem cells, cloning • Privacy & transparency NHGRI/DOE ELSI, Genetic Screening Study Group

Anonymity, privacy, disclosure, identity

"Open-source" meets Personal Genome-Phenome Project • Are information-rich resources (e. g. facial imaging & genome sequence) really anonymous? • What are the risks and benefits of "open-source"? • What level of training is needed to give informed consent on open-ended studies? • Harvard Medical School IRB Human Subjects protocol submitted 16 -Sep-2004.

Grand Challenges Are the hypervariable regions of HIV the best or the worst T-cell vaccine targets? 10, 000 peptides by 100 HLA types with Andrew Levin & Norm Letvin HIBIE Harvard Inst. Biologically Inspired Engineering Chips to DNA to cells to embryos . DNA lattices for polonies and synthetic arrays with Joe Jacobson, John Reif SETG (Search for Extra-Terrestrial Genomes (NASA) Microfluidic PCR With Gary Ruvkun, Mike Finney, Maria Zuber, Wally Gilbert Protein Design : Recombinase & Carbon Nanotube Synthase With David Baker Jun 04 -Mar 05 -present

Synthetics: A 100 km view 360 km ISS 1000 km inner van Allen belt 35, 920 km geosynchronous 384, 000 km to earth's moon 100 M km to Mars

From 100 km down to a 1 nm view Kim et al. (2003) Phys Rev Lett. 90: 065501. Dynamics of fullerene coalescence. Han et al. Phys. Rev. B (2004) Microscopic Mechanism of Fullerene Fusion. Pantarotto et al. (2004) Chem Commun. 2004 Jan 7; (1): 16 -7. Translocation of bioactive peptides across cell membranes by carbon nanotubes. Bachtold A, Hadley P, Nakanishi T, Dekker C. Science. 2001 Nov 9; 294(5545): 1317 -20. Logic circuits with carbon nanotube transistors.

Sequencing cost & imaging (30 to 100, 000 fold improvements) ABI # bp/expt Complexity (bp) Avg Fold Cov 8 Pix per bp Read-length 900 $ / kb (e<1 e-5) 2. 4 $/ 1 X 3 e 9 b 2 e 6 Indel Error 5 e-3 Subst Error 4 e-3 3 X Cons Err 1 e-4 Kb / min 0. 8 Pix / sec Enz $/mg - 2004 2 e 7 74 3 e 5 300 14 (SBE) 0. 6% 4 e-6 360 2 e 5 8 Jun 2005 3 e 7 4 e 6 6 1724 25 (pair). 08 2 e 5 1 e-3 1 e-6 27 2 e 6 8 2006 3 e 8 3 e 9 0. 1 333 35. 04 5 e 4 1 e-3 3 e-7 1 e 3 6 e 6 8 >2007 60 e 9 6 e 9 10 1 42 1 e-5 100 1 e-3 1 e-7 1 e 6 2 e 7 0. 4

Lattices Yeast 12 Mbp = 4 mm long => 2 mm square. Grid by oligo array 5 micron ablated by e-beam down to a 10 nm resolution. Y 1 Y 1 Y 1 X 2 X 3 X 4 X 5 Y 2 Y 2 Y 2 X 1 X 2 X 3 X 4 X 5 Y 3 Y 3 Y 3 X 1 X 2 X 3 X 4 X 5 Y 4 Y 4 Y 4 X 1 X 2 X 3 X 4 X 5 Y 5 Y 5 Y 5 Anchoring via triple stand poly. Py regions (no denaturation) every 5 microns would match sites every 15 kbp in the yeast genome (7 -mer)

Aptamers for Syn. Bio &Imaging • J Am Chem Soc. 2004 126: 9266 -70. Modular aptameric sensors. Stojanovic MN & Kolpashchikov DM. • Programmable ligand-controlled riboregulators of eukaryotic gene expression. Nat Biotechnol. 2005 23: 337 -43. Bayer TS, Smolke CD.

Neuroimaging Invariant visual representation by single neurons in the human brain. Nature. 2005 435: 1102 -7. Activation of the fusiform gyrus when individuals with autism spectrum disorder view faces. Neuroimage. 2004 22: 1141 -50. Hadjikhani N, Joseph RM, Snyder J, Chabris CF, Clark J, Steele S, Mc. Grath L, Vangel M, Aharon I, Feczko E, Harris GJ, Tager-Flusberg H.

Bridging the Rift Ecosys-genomics, human neuroimaging/genomics Seed funding: Mati Kochavi,

Grand Challenges • Infection-proofing Vaccines New genetic codes , chirality • $1 K Genome Bioweathermap • Low-senescence, low-cancer stem cells • Neuroimaging • Autofabrication from simple environmentally abundant components like raw minerals, air, water, complex CAD. • Space

Possible next steps • Consider "six impossible things before breakfast". • Dream. Brainstorm. • Discuss possible synergies with others in the lab & collaborators. • Can we thereby turn challenging tasks into simple ones? • Drill down into the details of what really stops us from achieving the grand challenges.