Constructionism: parts

Collaborators Past BiologicalGrants Present AnalyticTech Transfer Future SyntheticCourses Computational Citizenship

NRB 258Thu 11-Jul-2005 12:30-2ish PM

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?

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.

http://www.chez.com/cropcircles/Nazca.htm

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

3 Exponential technologies(synergistic)

Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

1E-3

1E-1

1E+1

1E+3

1E+5

1E+7

1E+9

1E+11

1E+13

1830 1850 1870 1890 1910 1930 1950 1970 1990 2010

urea

E.coli

B12

tRNA

operons

telegraph

Computation &Communication

(bits/sec~m$)

Synthesis (amu/project~M$)

Analysis(kamu~base/$) tRNA

Present http://arep.med.harvard.edu/gclab3.htm (MS: Kyriacos Leptos, Michael Chou, Dat Nguyen) (SynReg: 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) (SynBio: 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) (3DReg:

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

Daley Stem/germ-cell DNA methylation, cancer drug resistance

Struhl Human haematopoetic stem cell model

Mitra, Gottlieb, Sherley Polonies & Stem Cells

Chisholm,Polz,JGI Single-cell sequencing Prochlorococcus

Ruvkun Environmental microbes & Mars

Ausubel, Kolter, Lory Biofilms

Laub,Dekker 3D chromosome

Perrimon RNAi & Imaging

Wu Pairing & homologous recombination

Letvin T-cell recognition of HIV gag x MHC diversity

Jacobson rE.coli (re-engineered E.coli)

Shared interests http://arep.med.harvard.edu/gmc/collab.html

Seq: Ambergen, Helicos, Caliper, MJR, NEN, Agencourt . SysteMS: BeyondGenomics, ThermoFinnigan, GenomaticaSyn: Xeotron/Invitrogen/Atactic, Nimblegen, CodonDevices

Technology Transfer CRI GTC Agencourt APG CollabDiag OscientPC Beckmann-Coulter '61 '93 '98 GPC '03 '05

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).

http://arep.med.harvard.edu/gmc/tech.html

Biological

bases Prochlorococcus 1.7M Solar energy & CO2 fix

Caulobacter 4.0 M 3D chromosome & cell structure

Escherichia 4.8M Genetic codes

Saccharomyces 12.0M Regulatory codes

Mus 3.0 G Embryonic stem cells

Homo 3.0 G Cancer & personal genomics

Technological 73-74 Mycoplasma

74-77 Computational crystallography: CORELS, 1st folded-NA

77-78 DNA lattices & sequencing: 1st plasmid

78-80 RNA&DNAsplicing: ribozymes & meganucleases

80-85 Genomic sequencing: Ig-enhancers, 5mC

84-86 Embryonic stem cells

86-94 Multiplexing: 1st 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 3D 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, SysBio 03-05 SynBio 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-BioSpice 01-04 NHLBI-PGA (Seed/Seigo/Seidman) 02-07 DOE-GTL (04 SynBio supplement) 02-05 PhRMA 02-05 Stem cells (Zon) Computional 03-08 NIGMS-SysBio (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. & BioEngineering

Synthetic Biology

Minimal genomes

Synth chem & biomimetics

Metabolic engineering

Synthetic Genomics

Genetic engineering

Synthetic regulatory

circuits

Drug biosynth

Sensors

Energy

Materials

Vaccines

Analyticchem & omics

Synthetic proteomics

Systems biology &

protein design

Custom chip projects

Design Ordered Size When File Received Project

2003? Lindell 200K? 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 rE.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 CD44 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

Nimblegen

Xeotron

Combimatrix

analytic

&Quality

&Selection

http://arep.med.harvard.edu/web2/chips.htm

Engineering Biological Systems

Action Specificity %KO "Design"

Small molecules (drugs) sec Varies Varies Hard

Antibodies min Varies Varies Hard

RNAi hr Varies Medium OK

Riboregulators hr Varies Medium + /-

Insertion "traps" day Yes Varies Random

Recombination day Perfect Complete Easy

Proteasome targeting min Excellent Medium Easy

Physical environment sec-day

Microfabrication sec-day

DNA motif target & quantitative effects (via homologous recombination)

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 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 stop2. Delete RF1 (1 free codon, for new aa e.g. PEG-pAcPhe-hGH) 1. Codons: AGY Ser > UCX Ser2. tRNAs: AGY Ser > AGY Leu3. Codons: UUR/CUX Leu > AGY Ser4. tRNAs: UUR Leu > UUR Ser5. Codons: UCX Ser > UUR Ser (Leu & Ser now switched & 8 codons free)

Mirror world : enzyme, parasite, & predator resistance& access 2n diastereomers (n chiral atoms)

1. Transition mutants: EF-Tu, peptidyl transferase, pol/ligase with D-amino acids & L-r/dNTPs: Dedkova et al. Enhanced D-amino acid incorporation into protein by modified ribosomes. JACS 2003 125, 6616. Semizarov et al. Stereoisomers of dNTPs 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 mirror-image DNA. 1992 NAR 20:3325.

Mirror world :

http://www.chemgenes.com/ANP-8034.phpThymidine cyanoethyl di-isopropyl phosphoramidite-L(synthetic)-D (natural)

5 Mbp Genome assembly alternatives

1. cat

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 strainsvs. Random/simultaneous 1 or more stages

3. cat2. kan

Reppas & Church

Hierarchical de novo DNA assembly

Conditional Selectable genes: 2 = chloramphenicol, 3 = kanamycin (better: 2=supF, 3=taRNA)

Conditional Meganuclease sites: 4 = I-SceI taggg_ataa^cagggtaat 5 = I-DmoI gccttgccgg_gtaa^gttccggcgcg

Conditional Conjugative transfer elements:6 = ColE1 oriT 7 = F (incBCD) oriT

Condititional origins of replication (or in main chromosome oriC)8 = IncX ori-R6K (pir protein) 9 = IncPα oriV (trfA protein).

Hierarchical de novo DNA assembly

selection=2,3 cut sites=4,5 transfer=6,7 replication=8,9

84ab-2yz46 95-bc3yz57 84abc3yz46 95cd-3yz57 84-de2yz46 95cde2yz57 84abcde2yz46

95ef-3yz57 84-fg2yz46 95efg2yz57 84gh-2yz46 95-hi3yz57 84ghi3yz46 95efghi3yz57 84abcdefghi3yz46

84ij-2yz46 95-jk3yz57 84ijk3yz46 95kl-3yz57 84-lm2yz46 95klm2yz57 84ijklm3yz46

95mn-2yz57 84-no3yz46 95mno3yz57 84op-3yz46 95-pq2yz57 84opq2yz46 95mnopq2yz57 95ijklmnopq2yz57 84abcdefghijklmnopq2yz46

100kb 200kb 400 kb 800 kb 1.6Mbp

0

1

2

3

4

5

6

7

8

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

# of passages

Do

ub

lin

g t

ime

(h

r)

Q1

Q3

Q2-1

Q2-2

EcNR1

Sequence monitoring of evolution(anticipate escape & resistance)

Safer biology via synthetic biology• Systems modeling• HiFi 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

difficulty

Responsible Conduct In Research

http://www.nap.edu/readingroom/books/obas/

"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

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 iGEM: Genetically Engineered Machines

05 Bph 101 Genomics, Computing, Economics & Society

BMC-Bioinformatics (impact factor - 5.42)OmicsNature-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 typeswith Andrew Levin & Norm Letvin

HIBIE Harvard Inst. Biologically Inspired EngineeringChips to DNA to cells to embryos

DNA lattices for polonies and synthetic arrayswith Joe Jacobson, John Reif

SETG (Search for Extra-Terrestrial Genomes (NASA)Microfluidic PCRWith Gary Ruvkun, Mike Finney, Maria Zuber, Wally Gilbert

Protein Design : Recombinase & Carbon Nanotube SynthaseWith David Baker Jun04-Mar05-present

Synthetics:A 100km view

360 km ISS 1000 km inner van Allen belt 35,920 km geosynchronous

384,000 km to earth's moon100M km to Mars

From 100km down to a 1nm view

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.

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.

ABI 2004 Jun 2005 2006 >2007

# bp/expt - 2e7 3e7 3e8 60e9

Complexity (bp) - 74 4e6 3e9 6e9

Avg Fold Cov 8 3e5 6 0.1 10

Pix per bp - 300 1724 333 1Read-length 900 14 (SBE) 25 (pair) 35 42

$ / kb (e<1e-5) 2.4 - .08 .04 1e-5

$/ 1X 3e9 b 2e6 - 2e5 5e4 100

Indel Error 5e-3 0.6% 1e-3 1e-3 1e-3

Subst Error 4e-3 4e-6 1e-3 1e-3 1e-3

3X Cons Err 1e-4 - 1e-6 3e-7 1e-7

Kb / min 0.8 360 27 1e3 1e6

Pix / sec - 2e5 2e6 6e6 2e7Enz $/mg - 8 8 8 0.4

Sequencing cost & imaging(30 to 100,000 fold improvements)

Lattices Yeast 12 Mbp = 4 mm long => 2mm square. Grid by oligo array 5 micron ablated by e-beam down to a 10 nm resolution.

Y1 Y1 Y1 Y1 Y1X1 X2 X3 X4 X5 Y2 Y2 Y2 Y2 Y2X1 X2 X3 X4 X5 Y3 Y3 Y3 Y3 Y3X1 X2 X3 X4 X5 Y4 Y4 Y4 Y4 Y4X1 X2 X3 X4 X5 Y5 Y5 Y5 Y5 Y5

Anchoring via triple stand polyPy regions (no denaturation) every 5 microns would match sites every 15kbp in the yeast genome (7-mer)

Aptamers for SynBio &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

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, McGrath L, Vangel M, Aharon I, Feczko E, Harris GJ, Tager-Flusberg H.

Invariant visual representation by single neurons in the human brain. Nature. 2005 435:1102-7.

Bridging the Rift

Ecosys-genomics, human

neuroimaging/genomics

Seed funding: Mati Kochavi,

Grand Challenges • Infection-proofing Vaccines New genetic codes , chirality • $1K 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.