05Jul11_Lab - Church Lab

tentchoirΤεχνίτη Νοημοσύνη και Ρομποτική

15 Νοε 2013 (πριν από 3 χρόνια και 6 μήνες)

77 εμφανίσεις

Constructionism:
S

灡牴p


Collaborators Past Biological

Grants Present
Analytic

Tech Transfer Future
Synthetic

Courses
Computational


Citizenship


NRB 258

Thu 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, Genomatica

Syn:

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 & CO
2

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

Analytic

chem & 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
D
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 stop

2. Delete RF1

(1 free codon, for new aa e.g. PEG
-
pAcPhe
-
hGH)



1. Codons: AGY Ser > UCX Ser

2. tRNAs: AGY Ser > AGY Leu

3. Codons: UUR/CUX Leu > AGY Ser

4. tRNAs: UUR Leu > UUR Ser

5. 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/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.php

Thymidine cyanoethyl di
-
isopropyl phosphoramidite


b
-
L

⡳祮(桥瑩挩

b
-

⡮慴畲慬)

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 strains

vs. Random/simultaneous

1 or more stages


3. cat

2. 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


84a
b
-
2yz46

95
-
b
c3yz57 84ab
c
3yz46

95c
d
-
3yz57

84
-
d
e2yz46 95
c
de2yz57 84abcd
e
2yz46


95e
f
-
3yz57

84
-
f
g2yz46 95ef
g
2yz57

84g
h
-
2yz46

95
-
h
i3yz57 84
g
hi3yz46 95
e
fghi3yz57 84abcdefgh
i
3yz46


84i
j
-
2yz46

95
-
j
k3yz57 84ij
k
3yz46

95k
l
-
3yz57

84
-
l
m2yz46 95
k
lm2yz57 84ijkl
m
3yz46


95m
n
-
2yz57

84
-
n
o3yz46 95mn
o
3yz57

84o
p
-
3yz46

95
-
p
q2yz57 84
o
pq2yz46 95
m
nopq2yz57 95
i
jklmnopq2yz57 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
Doubling time (hr)
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)

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




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

1

Read
-
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
2e7

Enz $/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 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


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.