Thanks to:

.gov||||.edu||||.org||||.com|| ||

Read = = = = = = = = I/O = = = = = = = Write

1

25-Jan-2010 3-3:30 DOE  Session E: Synthetic BiologyGenome Engineering, Multi-virus Resistance & Accelerated Evolution for Industrial Chemicals (Harvard)

AzcoRBH

LSRF

2

(Moore’s law) 1.5x/yr for electronics

vs 10x/yr for

DNA Sequencing

4 logs in 4 years

2009:Lig:$1.5K

2005:capil:$50M

1995:gel: $3G

Pol:$50K

>20 years ahead of the 1970-2004 exponential

Seq bp/$ $/genome

3

1st-generationGene synthesis

vs2nd-generationSequencing & DNA synthesis

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

100

1000

10000

100000

1000000

1980 1990 2000 2010

dsDNAOligosSeq bp/$

Moore’s law =1.5x/yrvs 10x/yr 24Mb/$ 30kb/$

4

Ultra-low-cost sequencing1. Polonator SbL/P Open-source $170K device, haplotypes 2. CGI SbL $5K genome Rolony grid & 100Kb haplotypes3. Roche-454 SbP Long reads (>0.4 kb)4. Illumina-GA SbP Fluorescent read-length 2*110 bp5. AB-SOLiD SbL Longest ligation reads6. Helicos SbP-sm High parallelism & quantitation7. Halcyon EM-sm Long reads (>Mb), $100 8. Ion Torrent SbP small device9. Genizon BioSci SbH In situ sequencing10. LightSpeed SbL 16X higher density, >10X speed11. Intelligent Bio SbP Hexagonal grid12. Pacific Bio SbP-sm Long reads (>2.0 kb)13. Bionanomatrix SbP-sm Fluorescent mapping14. OxfordNanopore Pore-protein-sm small device15. Visigen SbP-sm Pol <> dNTP FRET 16. ZS Genetics EM-sm Iodine labels17. Nabsys Pore-sm small device18. GE Global SbP-sm 19. IBM Pore Si-sm small device 20. Electronic Biosci Pore-protein-sm

Polonator

Polonator

5

Open-architecture hardware, software, wetware

Polonator

$160K

2 billion beads per run

e.g.:

1981IBM PC

1998Google

Rich TerryFigure 4.6.1 Polonator instrument

A shared resource

6

Open-access hardware, software, wetware

Polonator

From Sequencer to Bio-Fab: Selective release of Synthetic sequences &/or cell sorting(FACS)

Digital micromirrors

Flow- cell2 billion beads or cells/run

Photo-labileimmobilization

7

Cell Chassis Choice

  E.coli S.cer Synec. M.genBiomass doubling       20 90 180 800

Chemical tolerance  + ++ + -

ss-MAGE                ++ ? ? ?

ds-recombination    + ++ + -

Industrial use        ++ ++ + -

8

Genome engineering 2000-2006Dupont: 1,3 Propanediol (7 years & $400M R&D)135 g/l at 3.5 g/l/h135 g/l at 3.5 g/l/h90% of theoretical yield from glucoseheoretical yield from glucose

27 changes to 4.6 Mbp E.coli6 genes up, 13 down, 8 foreign genes

GlycerolDAR1 GPP2

Glycerol-3-P

- NADH- NADH

3HPA

coB12 - NADPH

yqhDdhaB1-3 1,3 propanediol

Yeast Klebsiella E.coli

for Sorona polymers

9

Bio-petroleum from microbes flotation -- not distillation

aqueous

organic

0

10

20

30

40

50

60

70

80

90

100

Extracellular Intracellular

Localization

Dis

trib

utio

n (%

tota

l)

1 2 3 4

110

3,000

50

3 months

Gasoline & diesel for current engines &

infrastructure

Fatty acid derived

10

Algal, cyanobacterial & fungal pathways to triglycerides, alkanes, olefins, terpenes

Botryococcus brauniidecarbonylase

http://www.biofuelsdatabase.org/map/alkane-decarbonylation_map.shtml http://www.springerlink.com/content/p6451qx982638856/fulltext.pdf

Methylelcosene from Prasiola stipitata

Helioculture™ Technology

11

12,800 mols PAR photons

into SC

Joule Diesel Productivity Model- photon perspective

Extraterrestrial: 43,100 MJ Total Radiation 16,900 MJ PAR

Atmosphere, Diurnal Cycle

Phoenix: 3400 MJ PAR ≈ 15,100 mols PAR photons

Historical Average Total Radiation to the Ground(MJ/m2/year)

Historical Average PAR Radiation to the Ground(MJ/m2/year)

Lanai, HI 7120 3530

El Paso, TX 7460 3460

Phoenix, AZ 7300 3400

Las Vegas, NV 7190 3320

Albuquerque, NM 6990 3240

Leander, TX 6050 3000

85% Photons Transmitted into SC

Diesel: 15K gal/acre/yr

NREL insolation data

12

226 kJ/mol incident PAR photons

85% photon utilization

926 mols CO2 reduced

Unless otherwise stated, all values are /m2/year

880 mols CO2 reduced 10,900 mols PAR photons

11.75 photons per mol CO2 reduced

5% to cellular maintenance

835 mols CO2 reduced to Diesel= 70 mols Diesel produced (C12H26)

170 g/mol

5% of operation time to culture growth

11.8 kg Diesel

13

Existing Sensors (select for new ligands)54 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rhaS rpoE rpoH rpoN rpoS soxS torR trpR tyrR

12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine

http://pubs.acs.org/doi/abs/10.1021/ja048634j

14

Multiplex Automated Genome Engineering2 hr Cycle time. For optimization & multivirus resistance

HarrisWang

15

Multiplex Automated Genome Engineering (MAGE)

Wang HH, in preparation, 2009

Optimized Parameters• Oligo length: 90mer• Oligo 2ndary structure: <12 kcal/mol• Oligo half-life: 5’ phosphothiol bps• Oligo conc.: up to 50 uM

• Cycle time: 2 to 2.5 hrs• >30% efficiency per cycle

Allelic Replacement• Strain: MG1655, mutS, integrated -Red

• Highly complex oligo pools for multiplexed multi-loci modifications

• >4 billion bp of targeted genetic variation produced per day

16Wang H et al Nature 2009

5-fold improvement in 3 daysAND improve growth rates

Accelerated Evolution 23K combinations per gene Lycopene (hydrocarbon): 20 genes up, 4 down, 2 new

17

Genome Engineering strategies

1. Random mutagenesis limited by lethal rate2. Genome synthesis 3. Semi-random4. Cyclic semi-random (MAGE)5. Directed mutations (MAGE)

18

Improving process yield, health, safety with 314+

changes

What threatens all biological systems?

What do all viruses have in common? or lack?

19

PEG-pAcPhe-hGH (Ambrx) high serum stability314 TAG to TAA changes

IsaacsCharalelChurchSunWang CarrJacobsonKong Sterling

New translation code: novel amino acidsSafety features: no functional DNA exchange

multi-virus resistance

TTT

F

30362 TCT

S

11495 TAT

Y

21999 TGT

C

7048

TTC 22516 TCC 11720 TAC 16601 TGC 8816

TTA

L

18932 TCA 9783 TAASTOP

STOP

2703 TGA STOP 1256

TTG 18602 TCG 12166 TAG 314 TGG W 20683

CTT

L

15002 CCT

P

9559 CAT

H

17613 CGT

R

28382

CTC 15077 CCC 7485 CAC 13227 CGC 29898

CTA 5314 CCA 11471 CAA

Q

20888 CGA 4859

CTG 71553 CCG 31515 CAG 39188 CGG 7399

ATT

I

41309 ACT

T

12198 AAT

N

24159 AGT

S

11970

ATC 34178 ACC 31796 AAC 29385 AGC 21862

ATA 5967 ACA 9670 AAA

K

45687 AGA

R

2896

ATG M 37915 ACG 19624 AAG 14029 AGG 1692

GTT

V

24858 GCT

A

20762 GAT

D

43719 GGT

G

33622

GTC 20753 GCC 34695 GAC 25918 GGC 40285

GTA 14822 GCA 27418 GAA

E

53641 GGA 10893

GTG 35918 GCG 45741 GAG 24254 GGG 15090

1

2

3

4

20

Multi-virus resistance:stop codons: TAG / total

X-174 5,386 b ss-DNA 0 / 9M13 6,407 b ss-DNA 1 / 10MS2 3,569 b ss-RNA 2 / 4T7 39,937 b ds-DNA 6 / 60T4 168,903 b ds-DNA 19 / 277E.coli 4,639,675 b ds-DNA 314 /1,360,152

ncbi.nlm.nih.gov/nuccore/9626372 56718463 176120924 9627425 29366675 (7 tRNAs: RITSPGL)

FarrenIsaacs

21

400 tRNAs /sec20 aminoacids/sec

mRNA

tRNAs

50S/Ribosome

Elongation factors30S

22

Applications of in vitro translation•Ribosome display•Membrane protein drug receptor studies•Personal cancer vaccines.•Labeling one protein not the whole cell.•New chemistries (e.g. mirror chirality)

>11 Commercial Systems: Roche, Ambion, Novagen, Promega, Invitrogen, Qiagen, Stratagene, Paragon, Amersham,Sutro,NEB

Tony Forster(Vanderbilt)

23

Mirror world : 2 key chirality gatekeepers: peptidyl transferase & AA-tRNA synthetasesSo: 10 bp change in 23S rRNA + a 46 nt ribozyme

Goals: Multi-enzyme resistance, metabolic isolation, new chiral chemistries

24

Mirror world :

A highly flexible tRNAacylation method fornon-natural polypeptideSynthesis. Suga lab Nature Methods 2006

Construction of Modified Ribosomes forIncorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006

25

113 kbp DNA 151 genes

Pure translation:Forster & Church

MSB ’05 GenomeRes.’06

Shimizu, Ueda ’01

Not minimal:High speed &

accuracy requires a few extra genes(E.coli 20 min.

doubling)

Reconstituted ribosomes:

Jewett & Church

26

Ribosomes from synthetic 23S rRNA with coupled translation of active luciferase

Traub & Nomura 1968 reconstitution of 30S

Mike Jewett

Synthetic 23S rRNA(+) (-)

ng/ml

30SR50S

30S50S

30S-- 5S rRNA

TP5030SIVT 23S rRNA

5S rRNATP5030S --

25-Jan-2010 3-3:30 DOE  Session E: Synthetic BiologyGenome Engineering, Multi-virus Resistance & Accelerated Evolution for Industrial Chemicals

1. Polonator: Merge next-gen Seq & Synth2. LS9-Chevron & JouleBio hydrocarbon production 3. MAGE: combinatoric: Lycopene4. MAGE: 314 change for multi-virus resistance5. Synthetic ribosome replication

28

.