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