Thrust 3: Chasses Design & Characterization

Who DeliverablesTom Knight Simple Chasses (e.g. Mesoplasma florum)George Church Chasses, codes, & characterization Drew Endy Virtual Machine (orthogonal DNA,RNA,Protein)Adam Arkin Modeling Jay Keasling Artificial chromosomesKris Jones Prather Engineered Metabolism impact load

Technology Integration

Tumor-seekingmicrobe

Drug-producingmicrobe

system requirements

Future testbeds

Integration

Standards

Knowledge Base

Characterization

Composability

Design

Abstraction

Technology Base

Fundamental knowledge

interdependencies

Existing PDC

Other resources

Parts

DevicesChasses

• George Church• Duhee Bang• Nick Reppas• Resmi Charalel• Chris Brown• John Aach• MCB100 students

• Joe Jacobson• Jason Park• Tiffany Yu• Bram Sterling• Eitan Reich• Chris Emig• Dave Kong

Collaborators on rE.coli new code

Collaborators: Shuguang Zhang, Franco Cerrina, Jindong Tian, Codon Devices, Nimblegen, Agilent, Atactic/Xeotron

Quant. specs/measures/milestones: 4 goals, yr 2

Goal 1 – Components to be changed• Pathway removal (for more promoters) : # paths = 2• Cell heterogeneity (e.g., ara transporter) : variance x0.7• Code changes : #aa, # phage & resistance level x100• Introduce novel chemistries into cells : sup efficiency x1.5

Goal 2 – Chasses robust to change & minimal mutations• dam,dnaEQ,mutDHLMRSTY,oxyR,polAC,

recAG,ssb,topB,ung,uvrD,vsr : mutation rate x0.5• insertion elements & transposons : mutation rate x0.1

Goal 3 – Additional chromosome

• Isolate exogenous gene function from native chromosome : I/O transfer function for main/plasmid/BAC • Origin: Partition, Addiction : loss rate x0.3

Goal 4 – Safety controls on the chassis• Delete phage lysogens & receptors (e.g. LamB) • Delete surface toxins (LPS) : quant sepsis, innate imm.• Low conjugation (Express traS and traT) : escape rate x.1• Add complicated or rare auxotrophies to prevent survival

outside the lab (aTc-tetR, Dap) : t1/2 = 1 to 90h; escape % x0.01

• Remove all antibiotic resistance genes : MIC x0.1

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

rE.coli Strategy II: “top-down” recombination

red recombination

Hierarchical recombination-conjugation strategy

10 stages

*UAG UAA codon replacement

UAG UAA Recombinant Design

*UAG UAA codon replacement

1. LRH: Left Region of Homology + UAA mutation (Genomic)2. SIL: Safe Insertional Site Fragment + UAA mutation (Genomic)3. GSC: General Selectable Cassette (e.g., kan, cat) (Synthetic)4. RRH: Right Region of Homology (Genomic)

λ Red Recombination of Crossover PCR Products

1 - 5 6 - 10 11 - 14 1 - 5 6 - 10 11 - 151 2 – 12- 1516181618-C wt

Genomic-GenomicGenomic-Synthetic

Positive clones for relA gene: 5, 8, 9, 11

*UAG UAA~ 20 bp overlap

Tm~60C

Multiplexing Crossover PCRs

• Employ Similar Approach across ~300 UAG sites

– Use orthogonal sequences at crossover site (^) for selectable marker cassette

• Multiplex PCR amplifications in a single, or few, reactions

^ ^

UAG Codon Distribution

0

5000

10000

15000

20000

0 100 200 300

# codons

Dis

tan

ce

Release Factor 1 (RF1) Reversible Knockout

• hemA: Glutamyl-tRNA reductase catalyzes the first step of porphyrin biosynthesis

• prfA: peptide chain release factor RF1 (targets UAG & UAA)

• prmC: protein-(glutamine-N5) methyltransferase that shows activity toward polypeptide chain release factors RF1 and RF2

• Replace endogenous copy of RF1 (prfA) with tunable version– Interfere with translation of UAG-containing genes

Transcriptional Control

Transcriptional & Translational Control

Isaacs, et al. Nature Biotechnology 22 (2004)

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

Autocatalytic Chasses Improvements

1. Synthesize two parts - join - purify

2. Solid phase : deblock - join - wash

3. Yield of < 0.1% requires selection

4. 20%: 2 steps/mut to 2 mutations/step

5. 99% multiple mutations

6. Design & evolution for #5.

rE.coli Projects • 113 kbp mini-genomes ribosome-display selection

• 4.7 Mbp new genetic codes protein drugs • 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

rEcoded Ecoli

Why Sequencing?

Synthesis by SequencingSynthesis accuracy dependent on sequencing accuracy

Sequencing by Synthesis:Sequencing by Extension (SbE)Sequencing by Ligation (SbL)

Evolutionary optimization, (Ribo) Sensors, metagenomics

‘Next Generation’ Sequencing Technology Development

Multi-molecule Our roleABI/APG Seq by Ligation (SbL)454 LifeSci Paired ends, emulsionSolexa/Lynx Multiplexing & polonyCGI SbLAffymetrix Software

Single molecules Helicos Biosci SAB, cleavable fluorsAgilent Nanopores

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

• 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

Societal ImpactSafety – clinical, accidental, threat

Surveillance – consortium started with Drew Endy, Codon, Blue Heron, DNA2.0, etc.

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

.

Virtual Lab TourSite: Harvard Medical School ‘New Research Building’

SynBERC HMS NRB

room 2384000 sq ft

http://arep.med.harvard.edu/photo/NRB238.pdf

Offices

Offices

ColdRm

1

Desks

Desks

EquipRm

1

EquipRm

2

TissueCulture& PCR setup

Computer rm 1

Computer rm2

Benches

Chem hood 1

SynBERC HMS NRB

room 2322000 sq ft

http://arep.med.harvard.edu/photo/NRB232.pdf

Polony room

Cold room

Machine shop 1

Conf. room

Benches

Chem hood2

Desks

AutoclaveKitchen

SynBERC HMS

Racks for instrument prototypes

SynBERC HMS Polony

room:seven

sequencing- by-synthesis microscopes