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Harvard MIT DOE GtL Center. C.Ting. 2-20 μm. 7-Feb-2005 4:10-4:40 PM. Collaborating PIs: Chisholm, Polz, Church, Kolter, Ausubel, Lory arep.med.harvard.edu. 0.6 μm. Molecular Systems Biology Access is free of charge. Transcriptomics Proteomics Metabolomics Functional genomics

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Harvard mit doe gtl center

HarvardMIT DOEGtL Center

C.Ting

2-20 μm

7-Feb-2005 4:10-4:40 PM

Collaborating PIs:

Chisholm, Polz, Church, Kolter, Ausubel, Lory

arep.med.harvard.edu

0.6 μm


Molecular systems biology access is free of charge

Molecular Systems BiologyAccess is free of charge

Transcriptomics

Proteomics

Metabolomics

Functional genomics

Structural genomics

Computational biology

Theoretical biology

Mathematical biology

Synthetic biology

www.nature.com/msb/


Harvard mit doe center projects

Harvard MIT DOE Center Projects

arep.med.harvard.edu

Poster# Topic Goal#

2. Leptos, et al. Proteomics1

121. Nguyen, et al. Mass spectrometry XML1

122. Nguyen, et al. Gene Regulation2

67. Thompson, et al. Vibrio diversity3

68. Martiny, et al. Prochlorococcus diversity3

77. Sullivan, et al. Cyanophage diversity1,3

3. Zhang, et al. Single cell sequencing1-4

1. Church, et al. Metabolic fluxes4

ProchlorococcusPhotosynthesis, circadian & cell cycles

EscherichiaSynthetic genomes/proteomes

Vibrio4X faster replication than E.coli

CaulobacterAsymmetric cell & chromsome structure

PseudomonasBiofilms


Prochlorococcus 40 n 40 s

Prochlorococcus40ºN - 40ºS

Ocean chl a (Aug 1997 –Sept 2000)

Provided by the SeaWiFS Project, NASA


Energy co 2 sequestration

Energy & CO2 Sequestration

Humans consume 2kW per person = 1010 kW.

Sunlight hits the earth at 40,000 times that rate (70% ocean).

CO2 370 ppm = 730 x1015 g globally, increase ~3 x1015 /yr.

Ocean productivity = ~100 x1015 g CO2/yr … due to

Autotrophs: 1026 Prochlorococcus cells globally (108 per liter)

Sequestration v. respiration v. use: heterotrophs (Pelagibacter),

phages, predators (Maxillopoda, Malacostraca, herring)

0.1 mm

0.1 m

6 cm

http://www.gsfc.nasa.gov/gsfc/service/gallery/fact_sheets/earthsci/terra/earths_energy_balance.htm

http://clear.eawag.ch/models/optionenE.html http://en.wikipedia.org/wiki/Copepod

Morris et al. Nature 2002 Dec 19-26;420(6917):806-10.

http://hosting.uaa.alaska.edu/mhines/biol468/pages/carbon.html

http://www.aeiveos.com/~bradbury/Papers/PhotosyntheticEfficiency.html


Harvard mit doe gtl center

Diel (circadian) cycle

Light output for sun-box: 14hr light – 10hr dark, 230 mE at peak

Zinser, Lindell,Chisholm,

Leptos, Jaffe, Lin,et al.


Harvard mit doe gtl center

Diel Expression: All genes

Dark

Dark

Light

Light

Normalized expression

Time (Hours)

Zinser et al. unpubl.


Harvard mit doe gtl center

Light regulated Prochlorococcus metabolism

glgA

glgB

glgC

Central

Carbon

Metabol.

a-Glc-1P

ADP-Glc

glycogen

a-1,4-glucosyl-glucan

glgX

glgP

Zinser et al. unpubl.


Oxygenic photosynthesis

H

O

O

2

2

Oxygenic Photosynthesis

Core reaction Center Proteins

e

e

-

-

e

e

-

-

NADPH

NADPH

psbA=D1

Fd=

Ferridoxin

PSII

PSII

PSI

PSI

D2

Pc= Plastocyanin

HLIP=

High

Light

Induced Protein

H

H

O

O

O

O

2

2

2

2


Photosynthetic genes in phage

HLIP

D1

Photosynthetic Genes in Phage

Podovirus P-SSP746 kb

Myovirus P-SSM2255 kb

PC

PC

HLIPs

HLIPs

Fd

Fd

D1

D1

12kb 24kb

12kb 24kb

Myovirus P-SSM4 181 kb

HLIPs

HLIPs

D1

D1

D2

D2

~500

~500

bp

bp

6.4kb

6.4kb

2.8kb

2.8kb

Lindell, Sullivan, Chisholm et al. 2004


Rna responses to phage

RNA Responses to Phage

MED4 host

psbA

MED4-0682 (60 aa Conserved URF)

Phage SSP7

psbA

Lindell,Sullivan, Zinser, Chisholm


Synthetic homologous recombination testing of dna motifs

Synthetic - homologous recombination testing of DNA motifs

1.3 2.4 (1.3 in DargR)

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 Genome Res. 14:201–208


Synthetic genomes proteomes why

Synthetic Genomes&Proteomes. Why?

  • Test or engineer cis-DNA/RNA-elements

  • Access to any protein (complex) including

  • post-transcriptional modifications

  • Affinity agents for the above.

  • Protein design, vaccines, solubility screens

  • Utility of molecular biology DNA -- RNA -- Protein

  • in vitro "kits" (e.g. PCR -- T7 -- Roche)

  • Toward these goals design a chassis:

  • 115 kbp genome. 150 genes.

  • Nearly all 3D structures known.

  • Comprehensive functional data.


Pure translation utility

(PURE) translation utility

Removing tRNA-synthetases,

translational release-factors,

RNases & proteases

Allows:

Selection of scFvs[antibodies] specific for HBV DNA polymerase using ribosome display. Lee et al. 2004 J Immunol Methods. 284:147

Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353

High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al. 2004 Eur J Biochem 271:568

Cell-free translation reconstituted with purified components. Shimizu et al. 2001 Nat Biotechnol. 19:751-5.

Also: membrane incompatible expression & diverse amino-acids (>21)


In vitro genetic codes

yU

mS

eU

UUG

UGG

CAG

| | |

| | |

| | |

...

AUG

AAC

ACC

GUU

GAA

5'

A 3'

fM

N

T

V

E

in vitro genetic codes

5'

3'

Second base

A

U

A

C

U

C

A

C

yU

mS

U

G

eU

80% average yield

per unnatural coupling.

eU = 2-amino-4-pentenoic acid

yU = 2-amino-4-pentynoic acid

mS = O-methylserine

gS = O-GlcNAc–serine

bK = biotinyl-lysine

Forster, et al. (2003) PNAS 100:6353

Zhang et al. (2004) Science. 303:371


Oligos for 150 776 synthetic genes for e coli minigenome m mobile whole genome respectively

Oligos for 150 & 776 synthetic genes(for E.coli minigenome & M.mobile whole genome respectively)

Forster & Church


Up to 760k oligos chip 18 mbp for 700 raw 6 18k genes

Up to 760K Oligos/Chip18 Mbp for $700 raw (6-18K genes)

<1K Oxamer Electrolytic acid/base

8K Atactic/Xeotron/InvitrogenPhoto-Generated Acid

Sheng , Zhou, Gulari, Gao (U.Houston)

24K Agilent Ink-jet standard reagents

48K Febit

100K Metrigen

380K NimblegenPhotolabile 5'protection

Nuwaysir, Smith, Albert

Tian, Gong, Church


Improve dna synthesis cost

Improve DNA Synthesis Cost

Synthesis on chips in pools is 5000Xless expensive per oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!)

Solution: Amplify the oligos then release them.

10 50 10 => ss-70-mer (chip)

=> ds-90-mer

=> ds-50-mer

20-mer PCR primers with restriction sites at the 50mer junctions

Tian, Gong, Sheng , Zhou, Gulari, Gao, Church Nature 2004


Improve dna synthesis accuracy via mismatch selection

Improve DNA Synthesis Accuracyvia mismatch selection

Other mismatch methods: MutS (&H,L)

Tian & Church


Computer aided design polymerase assembly multiplexing cad pam

Computer Aided Design Polymerase Assembly Multiplexing (CAD-PAM)

50

75

125 225 425 825 … 100*2^(n-1)

Moving forward:

1. Tandem, inverted and dispersed repeats

(hierarchical assembly, size-selection and/or scaffolding)

2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates

3. 15kb to 5Mb by homologous recombination (Nick Reppas)

4. Phage integrase site-specific recombination, also for counters.

Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB.


All 30s ribosomal protein dnas codon re optimized

All 30S-Ribosomal-protein DNAs(codon re-optimized)

1.7 kb

0.3 kb

Atactic <4K chip

s19

0.3kb

Nimblegen 95K chip

Tian, Gong, Sheng , Zhou, Gulari, Gao, Church


Improving synthesis accuracy

Improving synthesis accuracy

Method Bp/error

Chip assembly (PAM) 160 1

Hybridization-selection 1,400 1

MutS-gel-shift 10,000 2

MutHLS cleavage 30,000 3

(10X better than PCR)

1. Tian, Church, et al. 2004 Nature 432:1050

2. Carr, Jacobson, et al. 2004 NAR 32:e162

3. Smith & Modrich 1997 PNAS 94:6847


Extreme mrna makeover for protein expression in vitro

Extreme mRNA makeoverfor protein expression in vitro

RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially.

RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable.

Solution:

Iteratively resynthesize

all mRNAs with less

mRNA structure.

Western blot based on His-tags

Tian & Church


Safe synthetic biology

Safe Synthetic Biology

Church, G.M. (2004) A synthetic biohazard non-proliferation proposal.

http://arep.med.harvard.edu/SBP/Church_Biohazard04c.doc

1. Monitor oligo synthesis via expansion of

Controlled substances, Select Agents, &/or Recombinant DNA

2. Computational tools are available; very small number of reagent, instrument & synthetic gene suppliers at present.

3. System modeling checks for synthetic biology projects

4. Multi-auxotroph, novel genetic code for the host genome, prevents functional transfer of DNA to other cells.


Harvard mit doe gtl center

Photosynthetic bacterial genomes

(for population genetics & proteomics)

89

97

92

71

66

100

78

72

84

0.01

MED4

TATL1a

high light adapted

Prochlorococcus

SAR6

ENATL1

ENATL3

MIT9302

MIT9312

MIT9201

GP2

MIT9202

MIT9215

TATL1b

MIT9107

NATL2A

low light adapted

Prochlorococcus

Pac 1

ENATL7

ENATL4

SS120

MIT9211

MIT9303

MIT9313

SAR139

WH8112

WH8102

SAR100

Marine Synechococcus

WH8101

WH8012

WH7805

SAR7

Synechococcus PCC6307


Harvard mit doe gtl center

Environmental population genomics(of a ribotype cluster)Thompson, Polz, et al. (2005) Science

Monthly

samples

Isolate

Vibrios

Identity population as

cluster of barcode genes

Quantification:

population is

continuously

present

Additional

marker gene:

highly

diverse

Genomes:

almost each

genome different

in typical sample


Sequencing single cells

Sequencing single cells

Biome studies focus on single-cells because hard to grow in the lab, multiple DNAs & RNAs per cell, exchange genome subsets.

(Complementary to Biome shotgun and/or 100 kb BACs)

Many input molecules required to sequence one molecule.

vs. one molecule sufficient to sequence via many copies of it.


Amplifying dna from single cells

Amplifying DNA from single cells

Prochlorococcus & Escherchia

Zhang, Martiny, Chisholm, Church, unpub.

No template control

f29 real-time amplification

Affymetrix quantitation of independent amplifications


Harvard mit doe gtl center

In vitro

libraries via

paired tag

manipulation

Monolayered immobilization in acrylamide

SOFTWARE

Images → Tag Sequences

Tag Sequences → Genome

Polony Bead Sequencing Pipeline

Bead polonies via

emulsion PCR

[Dre03]

Enrichment of amplified beads

FISSEQ or “wobble”

sequencing

Epifluorescence Scope with Integrated Flow Cell

Mitra, Shendure, Porreca, Rosenbaum, Church unpub.


Read length needs for population surveys

Read length needs for population surveys

Paired tags are separated by 1000 +/- 100 bases


Polony f luorescent i n s itu seq uencing libraries

Selector

bead

Polony Fluorescent In SituSequencing Libraries

1 to 100kb Genomic

2x20bp after MmeI (BceAI, AcuI)

LR

M

M

Sequencing

primers

PCR

bead

Greg Porreca

Abraham Rosenbaum

Dressman et al PNAS 2003 emulsion


Cleavable dntp fluorophore terminators

Cleavable dNTP-Fluorophore (& terminators)

Reduce

or

photo-

cleave

Mitra,RD, Shendure,J, Olejnik,J, Olejnik,EK, and Church,GM (2003) Fluorescent in situ Sequencing on Polymerase Colonies. Analyt. Biochem. 320:55-65


Harvard mit doe gtl center

Polony-FISSeq: up to 2 billion beads/slide

Cy5 primer (570nm) ; Cy3 dNTP (666nm)

Self Organizing Monolayer

Jay Shendure


High accuracy special case homopolymers e g aaa cc etc

High accuracy special case: homopolymers (e.g. AAA, CC, etc.)

  • Use "compressed" tags , ACG = ACCG=ACCCG

  • Quantitate incorporation

  • Reversible terminators

  • FRET between adjacent 3' bases

  • Wobble primers, CTAGCGAGCTAGNNNNNNNNA

    All five of these work.

  • Maintenance of amplification fidelity using linear amplification from initial genomic fragment


Harvard mit doe gtl center

Polony FISSeq Stats

  • # of bases sequenced (total)23,703,953

  • # bases sequenced (unique)73

  • Avg fold coverage324,711 X

  • Pixels used per bead (analysis)~3.6

  • Read Length per primer14-15 bp

  • Insertions 0.5%

  • Deletions 0.7%

  • Substitutions (raw) 4e-5

  • Throughput:360,000 bp/min

  • Current capillary sequencing 1400 bp/min

  • (600X speed/cost ratio, ~$5K/1X)

  • (This may omit: PCR , homopolymer, context errors)

Shendure


Wobble vs simple primer sequencing

Wobble vs Simple primer sequencing

1 vs 2.5 bp read/cycle of 4 bases

10 vs 14-200 bp reads

3e-3 vs 4e-5 non-homopolymer errors

3e-3 vs 1e-1 homopolymer errors

40 minutes per base tested =

60 hr per 20 cycles (20 hr, if 4 colors)


Harvard mit doe center projects1

Harvard MIT DOE Center Projects

arep.med.harvard.edu

Poster# Topic Goal#

1. Church, et al. Metabolic fluxes4

2. Leptos, et al. Proteomics1

68. Martiny, et al. Prochlorococcus diversity3

121. Nguyen, et al. Mass spectrometry XML1

122. Nguyen, et al. Gene Regulation2

77. Sullivan, et al. Cyanophages1,3

67. Thompson, et al. Vibrio diversity3

3. Zhang, et al. Single cell sequencing1-4

ProchlorococcusPhotosynthesis, circadian & cell cycles

EscherichiaSynthetic genomes/proteomes

Vibrio4X faster replication than E.coli

CaulobacterAsymmetric cell & chromsome structure

PseudomonasBiofilms


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