George Church Wed 20-Sep-2006 Science Center Hall E

Thanks to:

Synthetic, constructive,

Adaptive Biology

NHGRI Seq Tech 2004: Agencourt, 454, Microchip, 2005: Nanofluidics, Network, VisiGen Affymetrix, Helicos, Solexa-Lynx

iGEM International Genetically Engineered Machines : 37 teams

Harvard iGEM team 2006DNA NanostructuresTiffany Chan Katherine Fifer Valerie Lau Matthew Meisel CyanobacteriaHetmann Hsieh Jeffrey Lau Zhipeng Sun David Ramos Cell surface targetingPerry Tsai Lewis Hahn

Teaching FellowsChris Doucette Shawn Douglas Nicholas Stroustrup AdvisorsPamela Silver George Church Radhika Nagpal Jagesh V. Shah William Shih Alain Viel

http://openwetware.org/wiki/IGEM:Harvard/2006

The Central Dogma of Molecular Biology

Inheritance Messenger Sensors/structures/catalysts

Structures Sensors Inherited epigenetic states

(non-standard view)

DNA RNA Protein

Genetic code: UUU encodes Phe = F etcStop codons: UAG, UAA, UGA

iGEM project 1: Cyanobacterial Kai clock

24 hr cycle programmable time(works in vitro with

only 3 proteins)

2. DNA nanobox for controlled release of protein-pharmaceuticals

iGEM project 3:Cell surface targeting / bridging

3 Exponential technologies

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

DOE Biofuel Goals

Miscanthus v Panicum (switchgrass) 22 v 10 tons/haGoals: 2kg Hybrid seeds v 2 tons rhizomes

self-destruction to aid crop rotation, pretreatment$0.10/L goal (NEB >4, corn-EtOH:1.3 soy-diesel:1.93)

Integrated cellulases & fermentation to ethanol, butanol, biodiesel, alkanes $0.02/L

via metabolic engineering & lab evolution

Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, et al. Nat. Biotech 2003

Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Ro DK, et al. Nature. 2006 8

Programmable ligand-controlled riboregulators to monitor metabolites.

Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.

ON

ON

OFF

Genome & Metabolome Computer Aided Design (CAD)

4.7 Mbp new genetic codes new amino acids 7*7 * 4.7 Mbp mini-ecosystems biosensors, bioenergy, high secretors, DNA & metabolic isolation

•Top Design Utility, safety & scalability

CAD-PAM Synthesis (chip & error correction)

Combinatorics Evolution Sequence

How? 10 Mbp of oligos / $1000 chip

8K Atactic/Xeotron/Invitrogen

Photo-Generated Acid

Sheng , Zhou, Gulari, Gao (Houston)

12K Combimatrix Electrolytic

44K Agilent Ink-jet standard reagents

380K Nimblegen Photolabile 5'protection

Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997

PNAS

~1000X lower oligo costs

(= 2 E.coli genomes or 20 Mycoplasmas /chip)

Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction

Digital Micromirror Array

‘Next Generation’ Technology Development

Multi-molecule Our roleAB/APG/BEC Seq by Ligation (SbL)454/Roche Paired ends, emulsionSLXA/IVGN/NEB Multiplexing & poloniesCGI Seq by Ligation (SbL)Affymetrix Software

Single molecules Helicos Biosci SAB, cleavable fluorsPacific Biosci Advisor KPCBAgilent Nanopores Visigen Biotech AB

Smart therapeutics: Environmentally controlled invasion of cancer cells by engineered bacteria.

Anderson et al. J Mol Biol. 2006

Optical imaging: bacteria, viruses, and mammalian cells encoding light- emitting proteins reveal the locations of primary tumors & metastases in animals. Yu, et al. Anal. Bioanal. Chem. 2003.

accumulate in tumors at ratios in excess of 1000:1 compared with normal tissues. http://www.vionpharm.com/tapet_virulence.html

Regulated Capsule TonB, DapD

& new genetic code for safety

New in vivo genetic code: resistant to all viruses; novel amino acids

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 326 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

Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGRe.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability

IsaacsChurch

Forster

CarrJacobson

JahnzSchultz

1

2

3

4

To change the genetic code

1. Free up 1 or more codons by change 30 to 30,000 codons throughout the genome

2. Remove the RF or tRNA dedicated to those codon(s)3. Add orthogonal tRNA and synthetase protein4. Add selectable gene dependent on above

Schultz tRNAs

http://www.sciencemag.org/cgi/reprint/302/5645/584.pdf

Evolving orthogonal tRNA charging enzymes

http://schultz.scripps.edu/Images_103105/research_fig3.jpg

Microbial lab evolution

Lenski Citrate utilizationChurch Trp/Tyr exchangePalsson Glycerol utilizationEdwards Radiation resistanceIngram Lactate productionStephanopoulosEthanol resistanceMarliere ThermotoleranceJ&J Diarylquinoline

resistanceDuPont 1,3-propanediol production

Cross-feeding symbiotic systems:aphids & Buchnera

• obligate mutualism• nutritional interactions: amino acids and vitamins• established 200-250 million years ago• close relative of E. coli with tiny genome (618~641kb)

Aphids

Internal view of the aphid. (by T. Sasaki)

Bacteriocyte (Photo by T. Fukatsu)

Buchnera (Photo by M. Morioka)

http://buchnera.gsc.riken.go.jphttp://buchnera.gsc.riken.go.jp

Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).

trp/tyrA pair of genomes shows the best co-growth

Reppas, Lin & Church ; Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome(2005) Science 309:1728

SecondPassage

First Passage

Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes

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(optimize small molecule synthesis/transport)

Sequence trp-

Reppas, Lin & Church

Consensus error rate Total errors (E.coli)

(Human)

1E-4 Bermuda/Hapmap 500

600,000

4E-5 454 @40X 200 240,000

3E-7 Polony-SbL @6X 0 1800

1E-8 Goal for 2006 0 60

Goal of genotyping & resequencing Discovery of variantsE.g. cancer somatic mutations ~1E-6 (or lab evolved cells)

Why low error rates?

Also, effectively reduce (sub)genome target size by enrichment for exons or common SNPs to reduce cost & # false positives.

Position Type Gene LocationABI

ConfirmComments

986,334 T > G ompFPromoter-

10 Only in evolved strain

985,797 T > G ompF Glu > Ala Only in evolved strain

931,960 ▲8 bp lrp frameshift Only in evolved strain

3,957,960 C > T ppiC 5' UTR MG1655 heterogeneity

-3274 T > C cI Glu > Glu red heterogeneity

-9846 T > CORF6

1Lys > Gly red heterogeneity

Mutation Discovery in Engineered/Evolved E.coli

Shendure, Porreca, et al. (2005) Science 309:1728

• Glu-117 → Ala (in the pore)

• Charged residue known to affect pore size and selectivity

• Promoter mutation at position (-12)

• Makes -10 box more consensus-like

-12 -11 -10 -9 -8 -7 -6

AAAGAT

CAAGAT

Can increase import & export capability simultaneously

ompF - non-specific transport channel

3 independent lines of Trp/Tyr co-culture frozen.

OmpF: 42R-> G, L, C, 113 D->V, 117 E->APromoter: -12A->C, -35 C->ALrp: 1bp deletion, 9bp deletion, 8bp

deletion, IS2 insertion, R->L in DBD.

Heterogeneity within each time-point reflecting colony heterogeneity.

Co-evolution of mutual biosensorssequenced across time & within each time-point

.