Transcript Synthetic Biology

Constructionism: S parts
NRB 258
Thu 11-Jul-2005 12:30-2ish 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
• $1K Genome
Bioweathermap
• Low-senescence, low-cancer stem cells
• Neuroimaging
•Autofabrication
• Space
3 Exponential technologies
(synergistic)
Computation &
Communication
(bits/sec~m$)
1E+13
1E+11
1E+9
1E+7
1E+5
urea
1E+3
operons
Synthesis
(amu/project~M$)
tRNA
B12
1E+1
1E-1
E.coli
telegraph
1E-3
1830
1850
1870
Analysis
(kamu~base/$) tRNA
1890
1910
1930
1950
1970
1990
2010
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/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
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
3D chromosome
RNAi & Imaging
Pairing & homologous recombination
T-cell recognition of HIV gag x MHC diversity
rE.coli (re-engineered E.coli)
Technology Transfer
http://arep.med.harvard.edu/gmc/tech.html
CRI
'61
GTC
Agencourt
CollabDiag
OscientPC
'93
'98 GPC
'03
APG
Beckmann-Coulter
'05
Seq: Ambergen, Helicos, Caliper, MJR, NEN, Agencourt .
SysteMS: BeyondGenomics, ThermoFinnigan, Genomatica
Syn: Xeotron/Invitrogen/Atactic, Nimblegen, CodonDevices
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 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
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
http://arep.med.harvard.edu/web2/chips.htm
Nimblegen
Xeotron
Combimatrix
analytic
&Quality
&Selection
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
Engineering Biological Systems
Action Specificity
Small molecules (drugs)
sec
Varies
Antibodies
min
Varies
RNAi
hr
Varies
Riboregulators
hr
Varies
Insertion "traps"
day
Yes
Recombination
day
Perfect
Proteasome targeting
min
Excellent
Physical environment
sec-day
Microfabrication
sec-day
%KO "Design"
Varies Hard
Varies Hard
Medium OK
Medium + /Varies
Random
Complete Easy
Medium Easy
DNA motif target & quantitative effects
(via homologous recombination)
2.4 (1.3 in DargR)
1.3
1.1
1.3
2.5
0.7
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 stop
2. Delete RF1
(1 free codon, for new aa e.g. PEG-pAcPhe-hGH)
1.
2.
3.
4.
5.
Codons: AGY Ser > UCX Ser
tRNAs: AGY Ser > AGY Leu
Codons: UUR/CUX Leu > AGY Ser
tRNAs: UUR Leu > UUR Ser
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 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=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
95cd-3yz57
84-de2yz46
95ef-3yz57
84-fg2yz46
84gh-2yz46
95-hi3yz57
84ij-2yz46
95-jk3yz57
95kl-3yz57
84-lm2yz46
95mn-2yz57
84-no3yz46
84op-3yz46
95-pq2yz57
100kb
84abc3yz46
95cde2yz57
84abcde2yz46
95efg2yz57
84ghi3yz46
95efghi3yz57
84abcdefghi3yz46
84ijk3yz46
95klm2yz57
84ijklm3yz46
95mno3yz57
84opq2yz46
95mnopq2yz57
95ijklmnopq2yz57
84abcdefghijklmnopq2yz46
200kb
400 kb
800 kb
1.6Mbp
Sequence monitoring of evolution
(anticipate escape & resistance)
8
7
Doubling time (hr)
6
Q1
Q3
Q2-1
Q2-2
EcNR1
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
90 100 110 120 130 140 150
# of passages
Safer biology via
synthetic biology
difficulty
• 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
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 iGEM: 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 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 moon
100M km to Mars
From 100km down to a 1nm 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<1e-5)
2.4
$/ 1X 3e9 b
2e6
Indel Error
5e-3
Subst Error
4e-3
3X Cons Err
1e-4
Kb / min
0.8
Pix / sec
Enz $/mg
-
2004
2e7
74
3e5
300
14 (SBE)
0.6%
4e-6
360
2e5
8
Jun 2005
3e7
4e6
6
1724
25 (pair)
.08
2e5
1e-3
1e-3
1e-6
27
2e6
8
2006
3e8
3e9
0.1
333
35
.04
5e4
1e-3
1e-3
3e-7
1e3
6e6
8
>2007
60e9
6e9
10
1
42
1e-5
100
1e-3
1e-3
1e-7
1e6
2e7
0.4
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 Y1
X1 X2 X3 X4 X5
Y2 Y2 Y2 Y2 Y2
X1 X2 X3 X4 X5
Y3 Y3 Y3 Y3 Y3
X1 X2 X3 X4 X5
Y4 Y4 Y4 Y4 Y4
X1 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
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, McGrath 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
• $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.