Fabricating with DNA

George Church Wed 22-Aug-2007 9:15 9:30 AM4th Fab Lab Forum & Digital Fabrication SymposiumThanks to: Fabricating with DNAAppliedBiosystems, Helicos, Roche454, Illumina, CGI, IBS, Affymetrix, EnzymaticsPGP Volunteers & Donors !

Fab vs. Bio-fab+ Plays well with digital computers - No habla C++- Doesnt get DNA + DNA is its native digital media Needs us to replicate + We need them Needs expensive Fab (e.g. ICs) + Simple or complex inputs Intelligent Design + Evolution

Bio Inorganic interfaces Metal-oxide-semiconductors (silicateins for Ti & Ga oxides)

Magnetic components (magnetosomes in magnetotactic bacteria)

Optical fibers & lenses (e.g. venus basket sponge)

Bacterial reduction of salts to metals (e.g. Se, Au, Ag)

DNA origami -- highly predictable 3D nanostructuresDNA-nanotube-induced alignment of membraneproteins for NMR structure determinationRothemundNature06

Douglas, et al. PNAS07

Open-source hardware, software, wetware, ELSwarefor DNA reading & writing A 97-99Greg PoreccaRob MitraB 02-03 JayShendureMJR slide-cyclerGSI Microarray scanner78-84

87-97Church, Gilbert Genomic Sequencing (electrotransfer, chemiluminescence, film scanner)Higgins, Richerich, Auger, Smith Multiplex SequencingPolony

Open-source hardware, etc: Personal Genome ProjectD05 = C03 + fluidics Rich TerryE07Faster XYZF07 : $106K (down from $500K)including computer

G07: internal autosampler

10 Mbp of DNA / $300 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents380K Nimblegen/GA Photolabile 5'protectionTian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNASSpatially patterned chemistryAmplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction

Synthetic Biology: augmentation & combinatorics (not minimization)Synthetic DNA: 1Mbp per month (Codon Devices)New polymers in vitro affinity selection (Vanderbilt)Hydrocarbon & other chemical syntheses in E.coli (LS9) Bacterial & stem cell therapies (SynBERC & MGH) New codes: Viral resistant cells & new aminoacids (MIT)Synthetic Ecosystems Evolve secretion & signalingInterfaces of Genomics & Society Hierarchical, modular, evolvable

Mirror world: resistant to enzymes, parasites, predatorsMirror aptamers, ribozymes, etc. require mirror polymerases 834 AA Thermus Polymerase 834 AA352 AA Dpo4 Sulfolobus DNA polymerase IV 174 AA DNA Polymerase X African Swine Fever Virus. 2004: Bang & Kent: A One-Pot Total Synthesis of Crambin (46-mer)

2006: Torbeev &Kent: A 203-mer Covalent Dimer HIV-1 Protease.

Why synthesize (minimal) in vitro self-replication? Molecular biology kits:PCR, T7 RNA pol, in vitro translation.

Production of devices larger than or toxic to cells.

Directed evolution of drugs & affinity agents.

Mirror-image proteins

Tony Forster(Vanderbilt)Duhee Bang (HMS)

113 kbp DNA 151 genes

ideal for comprehensiveatomic, ODE & stochastic models

Forster & Church MSB 05 GenomeRes.06Shimizu, Ueda et al 01 Pure in vitro translating & replicating system

Genome engineering CAD70b 15Kb 5Mb 250 Mb Polymerase in vitroIsaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, ChurchRecombination in vivo E.coliError CorrectionMutS 1E-4Recombination in human cellsBacterial (Artificial) ChromosomesBACsHuman(Artificial) ChromosomesHACsSequencing 1E-7Chemical Synthesis1E-2

rE.coli Strategy #3: ss-Oligonucleotide Repair Obtain 25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, dam)Ellis et al. PNAS 2001Constantino & Court. PNAS 2003DNA Replication Fork

Multiplex Automated Genome Engineering (MAGE)Wash with water & DNA pool (50)Concentrate, electroporateResuspend, bubble, selectO-ringmembraneConcentrate Wang, Isaacs, Terry

Multiplex Automated Genome Engineering (MAGE)syringe pumpelectrically actuated valveselectroporation cuvette w/ membrane filterOD sensordata acquisition systemcomputer communication /Wang, Isaacs, Terry

Recombination-Cycling UAG to UAA E.coli Essential GenesMutation Distribution: 11 oligos, 15 cyclesMutation Distribution: 54 oligos, 45 cycles* Continuous cycling Scaling & Automation Increase Efficiency of RecombinationWang, Isaacs, Carr, Jacobson, Church

Chart5

4

11

12

19

20

7

3

1

Frequency

# mutations/clone

Frequency

E5025B20_Threshold at 1.5

1batop1babot1bbbtop1bbbbot1bcctop1bccbot1bdtop1bdbot1betop1bebot1bftop1bfbot

000000000000000000000000000000000000000000000000000000000000100000000000000000000010001000000000

100000011110000000000000011100000100000000000000011000000110000010010000000000000101000001000000

000000000000000010011000000000100000000000011010000100001110010010011000101001000001011000000001

000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000

000000000100000000000000010000000000000011100000000001001000000000010000010001000000010000000000

000000000000100100011001110000000000100100100101010001111010110111110011101010010000011101001000

001001001000000000000000001111000000000000000000001000000000001000000010000000001000000110000000

100000000000000000101000000010000000000000000000000000000100010010000000100000000000000000000010

000110001000001010010010000000000000011010000000000000001000000000000001001000000000000100000000

001000010000110000000000000010010000000000100010100000000001010000000001000100000010000000000000

110100010000000101001100100000010000000110000000011101011000000001000010000000011000000000001000

312211033210211221134111232231120100111231311121133203125331241142141033313112022122033312002011

BinFrequency

01212.765957446830.03

13638.2978723404180.19

22324.4680851064220.23

32021.276595744700.00

444.2553191489110.11

511.0638297872360.38

130.14

90.09

140.15

84140.15

230.24

E5025B20_Threshold at 1.5

0

0

0

0

0

0

# of mutations

% of population

Mutation Distribution in Population (N=50, R=20)

E5025B20_Threshold at 2

0

0

0

0

0

0

0

0

0

0

0

Loci

Frequency of Recombination

Recombination Frequency at various loci (N=50, R=20)

E5025B20_Manual

1batop1babot1bbbtop1bbbbot1bcctop1bccbot1bdtop1bdbot1betop1bebot1bftop1bfbot

000000000000000000000000000000000000000000000000000000000000100000000000000000000010001000000000

100000011110000000000000011100000100000000000000011000000110000010010000000000000101000001000000

000000000000000010011000000000100000000000011010000100001110010010011000101001000001011000000001

000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000

000000000100000000000000010000000000000011100000000001001000000000010000010001000000010000000000

000000000000100100011001110000000000100100100101010001111010110111110011101010010000011101001000

001000000000000000000000001111000000000000000000001000000000001000000000000000000000000000000000

000000000000000000100000000000000000000000000000000000000100000000000000100000000000000000000000

000110001000001010010010000000000000011010000000000000001000000000000001001000000000000100000000

001000000000010000000000000010000000000000100010100000000001010000000001000100000010000000000000

000000000000000101000000000000000000000100000000010001000000000000000010000000001000000000000000

102110012210111221132011132221100100111221311121132103114331231131141023313112011122033202001001

BinFrequency30.03

01819.1489361702

14244.6808510638180.19

22021.2765957447220.23

31414.893617021300.00

422.1276595745110.11

More00360.38

70.07

30.03

140.15

110.11

70.07

E5025B20_Manual

0

0

0

0

0

0

# of mutations

% of population

Mutation Distribution in Population (N=50, R=20)

Sheet2

0

0

0

0

0

0

0

0

0

0

0

0

Loci

Frequency of Recombination

Recombination Frequency at various loci (N=50, R=20)

Sheet3

20070124a_1b - Top20070124a_1b - Bottom20070124bb_1b - Top20070124b_1b - Bottom20070124c_1b - Top20070124cc_1b - Bottom20070124c_1b - Top20070124d_1b - Bottom20070124e_1b - Top20070124e_1b - Bottom20070124f_1b - Top20070124f_1b - Bottom

123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516

UAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RF

lolA100010101010101010100lolA100010101010101010100lolA100010101010101010100lolA1000101010100.90.11010100.1lolA100010101010101010100lolA100010101010101010100lolA100010101010101010100lolA100010101010011010101lolA100010101010101010100lolA100010101010101010100lolA100010100110101001102lolA100010101010101010100

ybjR9000.20.80.90.1100.90.10.90.10.90.110012.2ybjR9000.10.90.10.90.10.9101010100.90.12.8ybjR9000.10.90.90.10.90.10.90.10.90.10.90.1100.90.11.5ybjR9000.90.10.10.90.10.90.10.90.90.10.90.10.90.10.90.13.2ybjR9000.80.20.10.90.90.10.90.10.90.10.90.1100.90.11.6ybjR9000.90.10.80.20.90.10.80.20.90.10.90.10.90.10.90.11ybjR9000.90.1010.10.90.90.10.50.50.90.10.90.10.90.12.9ybjR9000.90.10.10.90.10.90.90.10.90.10.90.10.90.10.90.12.4ybjR9000.10.90.90.10.90.10.10.90.90.10.90.110102.2ybjR9000.90.10.90.10.90.10.90.10.90.10.90.110100.6ybjR900100.10.90.90.10.10.90.90.1100.90.10.90.12.2ybjR900100.10.90.90.110101010101

ybjK80010101010101010100ybjK80010011010101010101ybjK80001101001011010103ybjK80010101010101010011ybjK80010100.10.910101010100.9ybjK80010101001011001103ybjK80010101001101010101ybjK80001010110100110104ybjK80001101001011010103ybjK80001100110100110103ybjK80010101001100101103ybjK80010101010101010011

sucB7000101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100

ybcF60010101010101010100ybcF600100.30.71010101010011.7ybcF60010101010101010100ybcF60010011010101010101ybcF60010101010101010100ybcF60001010110101010103ybcF60010101010100110101ybcF60001101010101010101ybcF60010101001101010101ybcF60010011010100110102ybcF60010101010100110101ybcF60010101010101010100

priC5001001010101010100priC500101010100110100.50.51.5priC5000.10.9101001011010013.9priC50001011010101010102priC50010101010011010012priC500100.90.10110100110013.1priC50010011010100101014priC50001100110010110015priC50001010101101001016priC50001100110011010014priC50010101010100101013priC50010011010011010102

ybaA4000.70.30.50.50.70.30.40.60.60.40.30.70.70.30.70.33.4ybaA4000.40.60.80.20.70.30.60.40.40.60.60.40.70.30.90.12.9ybaA4000.80.20.50.50.50.50.50.50.50.50.50.50.80.20.60.43.3ybaA4000.50.50.60.40.50.50.40.60.70.30.60.40.50.50.60.43.6ybaA4000.50.50.40.60.50.50.50.50.50.50.60.40.40.60.70.33.9ybaA4000.60.40.40.60.50.50.50.50.50.50.50.50.60.4103.4ybaA4000.50.50.50.50.50.50.60.40.40.60.60.40.60.40.60.43.7ybaA4000.50.50.60.40.60.40.70.30.50.50.70.30.70.30.60.43.1ybaA4000.70.30.50.50.30.70.40.60.50.50.50.50.50.50.70.33.9ybaA4000.30.70.70.30.60.40.60.40.60.40.70.30.80.20.80.22.9ybaA400100.50.50.50.50.50.50.50.50.50.50.50.50.50.53.5ybaA400100.50.50.50.50.50.50.70.30.70.30.70.30.60.42.8

Lon3000.10.90.90.10.90.10.80.20.80.20.90.1100.90.11.7Lon30010101010100.90.110100.1Lon3000.20.80.90.10.10.90.90.10.10.90.90.1100.90.13Lon3000.90.10.90.10.90.10.90.10.20.80.90.10.90.10.90.11.5Lon3000.90.10.90.10.90.10.90.10.90.10.90.1100.90.10.7Lon3000.90.10.90.10.90.10.90.10.90.10.90.10.90.10.70.31Lon3000.90.10.90.10.90.10.90.10.90.10.90.10.90.10.90.10.8Lon3000.90.10.10.90.90.1100.90.10.20.80.90.10.90.12.2Lon3000.20.80.90.10.90.10.90.10.90.10.90.10.90.10.90.11.5Lon3000.20.80.90.10.90.1100.90.11010101.1Lon300100.90.10.90.10.90.10.90.10.90.10.90.10.90.10.7Lon300100.90.10.90.11010100.20.8101

pgpA20010101001011010102pgpA200010.30.71010101001102.7pgpA2000110100110100.30.7102.7pgpA20010101010101010100pgpA20010101010100101102pgpA20001101010101010101pgpA20010101010101010100pgpA20001101010101010101pgpA20010101010101010011pgpA20010100110101010101pgpA20010101010101010011pgpA20010101010101010100

yaiS150100.80.20.30.70.60.40.70.30.50.510013.1yaiS150101010100.10.90.10.9100.60.42.2yaiS150100.90.10.90.10.90.10.90.11010100.4yaiS15010100.90.10.90.10.10.90.90.10.90.1101.3yaiS1500.80.20.90.10.80.20.80.20.90.10.30.710101.5yaiS1500.90.1100.10.91010100.10.9101.9yaiS150010.60.40.90.10.90.10.70.31010101.9yaiS1500.80.210100.10.9100.20.810101.9yaiS1500.90.10.90.10.70.30.90.11010100.20.81.4yaiS1501010100.20.8101010100.8yaiS150100.80.20.10.90.90.11010100.90.11.3yaiS15010101010101010100

frmR1000.20.80.20.80.80.20.20.80.80.20.60.40.90.10.20.84.1frmR1000.90.10.40.60.90.10.90.10.40.60.20.8100.20.83.1frmR1000.90.10.20.80.70.30.70.30.20.80.20.80.50.50.80.23.8frmR1000.10.90.80.20.70.30.60.40.70.30.80.20.80.2102.5frmR1000.70.30.80.20.60.40.70.30.70.30.80.20.90.10.20.82.6frmR1000.20.80.60.40.90.10.70.30.70.30.80.20.90.1102.2frmR1000.70.3010.20.80.20.80.80.20.30.70.90.10.20.84.7frmR1000.20.80.70.30.30.70.90.10.70.30.90.10.90.10.90.12.5frmR1000.80.20.20.80.60.40.80.20.80.20.80.20.90.10.90.12.2frmR1000.90.10.80.20.30.70.80.20.90.110100.30.72frmR100100.70.30.20.80.80.20.80.20.90.10.90.10.90.11.8frmR100100.80.20.80.20.90.10.40.60.90.10.90.10.90.11.4

kankankankan0.90.1kankankankankankankankan

7.22.88.31.79.71.37.93.18.82.29.21.810.60.47.83.2Mut/clone8.42.66.94.19.71.310.50.57.93.18.82.29.71.38.12.9Mut/clone6.14.99.41.69.11.96.94.16.64.49.51.59.61.49.21.8Mut/clone8.42.67.43.69.11.98.92.18.52.510.10.910110.31.7Mut/clone9.71.39.11.98.82.29.81.28.92.18.52.59.31.78.72.3Mut/clone7.53.58.62.47.33.78.92.1929.11.98.42.69.61.4Mut/clone92748.62.48.52.59.31.77.73.39.31.78.62.4Mut/clone5.35.77.53.56.94.19.61.4836.94.110.40.69.31.7Mut/clone6.74.38.42.68.42.66.14.99.11.910.10.99.31.77.73.3Mut/clone7.33.79.31.76.74.39.51.59.31.78.62.410.80.29.11.9Mut/clone110927.63.48.22.810.10.97.33.77.23.88.12.9Mut/clone1108.32.710.10.910.40.69.11.910.60.49.81.29.51.5Mut/clone

CALLUAA321312031.875341032122511341112.125231120011.25011012121313111211.625132203121.75534124012.5322400131.875314112021.75013203321.7502002010.625

32131203341032125113411123112001011012123131112113220312534124013224001331411202013203320200201

16.51821.616.215.219.62024.122.217.419.59.2219.5Total # of mutations (11 loci)

2.335106383Avg mutations/clone

BinFrequency11.6755319149Avg mutations for 55 oligos

01512

13136

22123

31820

464

521

Sheet3

# mutations/clone

Frequency

54 Oligo Pool Distribution (11 loci assayed)

Sheet4

Sheet4

15

31

21

18

6

2

# mutations/clone

Frequency

E2506B

4

11

12

19

20

7

3

1

Frequency

# mutations/clone

Frequency

Sheet1

E2512B15

BinFrequency

04

111

212

319

420

57

63

71

total clones77

E2512B15

Frequency

# mutations/clone

Frequency

11 Oligo Pool Distribution

20070122a_1b - Top20070122a_1b - Bottom20070122b(a)_1b - Top20070120b(a)_1b - Bottom20070120b2(b)_1b - Top20070122b2(b)_1b - Bottom20070122c_1b - Top20070124d_1b - Bottom20070122d_1b - Top20070122d_1b - Bottom20070120e_1b - Top20070120f_1b - Bottom20070120f_1b - Bottom

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UAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RF

lolA1000101010101010100.10.90.9lolA100010101010101010100lolA1000101010101010100.10.90.9lolA100010101010101010100lolA100010101010101010100lolA10001010100.10.90.20.81010101.7lolA100010101010011001102lolA100010101010101001101lolA100010101010011001102lolA100010101010101010100lolA100010010110101010102lolA100010101010101010100lolA1000100110101101001010110100

ybjR90010101010100.90.1100.90.10.2ybjR9001010100.20.80.90.11010100.9ybjR900101010101010100.90.10.1ybjR900100.90.110100.90.11010100.2ybjR9000.10.91010100.10.9100.80.20.90.12.1ybjR900100.10.9100.30.70.30.71010102.3ybjR9001010100.90.1101010100.1ybjR9001010100.20.8100.20.80.90.1101.7ybjR90010101010101010100ybjR9001010100.90.1100.10.901102ybjR900100.90.1101010100.10.90.10.91.9ybjR90010101010101010100ybjR9001010100.10.90.9101010011010100

ybjK80001011010010101105ybjK80001011001010110105ybjK80001011010010101105ybjK80001011010010110104ybjK80001101010011010102ybjK80010011001011010103ybjK80010101010101001101ybjK80001101010101010101ybjK80010101010101001101ybjK80001101010101010101ybjK80010101010101010100ybjK80010101010101010100ybjK800101010100100110101010010

sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB700101010100101010101010100

ybcF60010011010100101014ybcF60001101010100110102ybcF60010011010100101014ybcF60001101010100110102ybcF60010101010101010100ybcF60001101010011010102ybcF60010101010011010101ybcF60010101010101001012ybcF60010101010011010101ybcF60010101010101001012ybcF60001101010100110102ybcF60010101010101010100ybcF600010101103101010101001010

priC50010011010010110014priC50010101010101010100priC50010011010100110013priC50010101010101010100priC500101010101010100.60.40.4priC50001101010101010101priC50010101001101010101priC50010101010101001101priC50010101010101010100priC50010101010101010100priC50010101010011010101priC50010101010101010100priC500100110101101010101001100

ybaA4000.90.10.60.410100.50.50.70.30.30.70.30.72.7ybaA400100.30.7100.80.20.20.81010101.7ybaA4000.90.10.60.41010100.80.20.20.80.30.72.2ybaA400100.30.710100.30.71010101.4ybaA4000.50.51010100.60.4100.60.4101.3ybaA4000.90.10.90.1100.90.10.80.21010100.5ybaA400100.90.1100.90.10.50.5100.30.70.80.21.6ybaA4000.90.10.40.6100.30.7100.30.70.80.20.40.62.9ybaA400101010100.60.4100.30.7101.1ybaA4000.90.10.30.7100.30.7100.40.60.90.10.30.72.9ybaA400100.20.80.20.80.50.50.60.40.70.30.50.5103.3ybaA4000.40.6101010101010100.6ybaA4000.20.80.40.60.70.30.80.21.9100.70.30.30.70.30.70.30.70.50.5100

Lon3000.10.91010101010100.20.81.7Lon3001010100.80.2101010100.2Lon3000.10.91010101010100.20.81.7Lon30010101010101010100Lon3000.10.91010100.10.91010101.8Lon300011010100.10.91010101.9Lon3001010101010100.10.9100.9Lon3000.10.91010101010010.10.92.8Lon3001010101010100.20.8100.8Lon3000.20.8101010101001012.8Lon30010101001101010012Lon30010101010101010100Lon3001010100.10.90.9100.10.910101001100

pgpA20001101010101010101pgpA20001101010100110102pgpA20001101010101010101pgpA20001101010100110102pgpA20010101010101010100pgpA20010101001011010102pgpA20010101010101010100pgpA20001100101101001104pgpA20010101010101010100pgpA20001101001101001103pgpA20010101010101010100pgpA20010101010101010100pgpA200010101103101010100110100

yaiS1500.20.8101010101010011.8yaiS15010101010101010100yaiS15010101010101010011yaiS15010101010101010100yaiS15010101010011010101yaiS15010101010101010100yaiS15010101010101001101yaiS15010101010101010100yaiS15010101010101010100yaiS15010101010101010100yaiS15010101010101010100yaiS15010101010101010100yaiS150101010100101010101010100

frmR10010011010010101104frmR10001011001011010104frmR10001011010100101104frmR10010011010010110103frmR10001101010011001014frmR10001101001011010103frmR10010011010010101104frmR10001011010101010013frmR10010101010011001102frmR10001011010101010013frmR10010101001100110013frmR10010101010101010100frmR100101010100100110101010010

kankankankan0.90.1kankankankankankankankankan

7.23.86.64.41121107.53.56.647.33.75.55.5Mut/clone748.32.71127.83.28.12.984110110Mut/clone746.64.41101101016.84.27.23.85.55.5Mut/clone838.22.81101108.22.87411011.90.1Mut/clone6.74.31101101105.85.21109.41.69.51.5Mut/clone6.94.1921106.34.74.46.6110110110Mut/clone1109.91.11109.81.27.53.51015.45.610.80.2Mut/clone749.41.61018.52.51109.51.55.75.37.53.5Mut/clone1101101101107.63.41106.54.5110Mut/clone7.13.99.31.71109.21.81109.51.56.94.17.33.7Mut/clone1019.11.99.21.88.52.59.61.48.72.39.61.48.12.9Mut/clone10.40.6110110110110110110110Mut/clone8.22.86.44.68.72.392Mut/clone1107.83.29.31.78.32.78.32.77.53.583Mut/clone

CALLUAA4434464.16666666674333454.44414463.833333333333343.2545133.25425703.601131601.71428571434213543.16666666673502.666666666742212442.7142857143122212132.815443.333333333335223C3233332.8333333333

4434464333454414463334451342570011316042135435042212442.71428571431222121315443522323333

BinFrequency

04

111

212

319

420

57

63

71

total clones77

BinFrequency

033

112

210

312

416

59

63

71

More0

33

12

10

12

16

9

3

1

0

4

11

12

19

20

7

3

1

Frequency

# mutations/clone

Frequency

11 Oligo Pool Distribution

Real dataSimulation

# roundsoligo %mean98-tilemean98-tile

E5025B20200.756.9166.9143.7778218607

40132110.5625

E1225B15152.53.0667

2047

20070124a_1b - Top20070124a_1b - Bottom20070124bb_1b - Top20070120b_1b - Bottom20070120c_1b - Top20070124cc_1b - Bottom20070120d_1b - Top20070124d_1b - Bottom20070124d_1b - Top20070124e_1b - Bottom20070120f_1b - Top20070120f_1b - Bottom

123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516123456789101112131415161234567891011121314151612345678910111213141516

UAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RFUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAAUAGUAALoci_RF

lolA100010101010100110101lolA10000.90.1101010100.10.910101lolA10001001101010100.70.3101.3lolA100010101010101010100lolA100001101010101010101lolA100001100110100101104lolA10000.20.8101010101010100.8lolA10000.10.9100.10.9100.10.91010102.7lolA1000100110100.50.5100.10.9102.4lolA100001101010101010101lolA1000101010100.10.91010100.9lolA100010101010101010100

ybjR9000.90.11010100.90.11010100.2ybjR9000.70.3100.90.10.80.20.80.20.20.8100.30.72.3ybjR9000.10.90.70.3101010100.90.10.80.21.5ybjR90010100.10.9100.90.110100.30.71.7ybjR9000.80.20.90.11010100.60.4100.20.81.5ybjR9000.20.80.90.1100.90.10.80.210010.90.12.3ybjR9001010100.50.5101010100.5ybjR9000.10.90.10.90.20.8100.90.110100.20.83.5ybjR9000.90.1010.10.91010100.30.7102.7ybjR9001010100.30.7101010100.7ybjR9000.90.110100.30.70.20.810100.80.21.8ybjR900101010100.20.80.80.2100.90.11.1

ybjK80001101001101010013ybjK80010100110100.10.910101.9ybjK80010011010101010012ybjK80010101010011010012ybjK80001011010101010102ybjK80010101001010101104ybjK80010101010101010100ybjK80010010110101010013ybjK80001101010101010101ybjK80010101010101010011ybjK80010101010011010012ybjK80010101010010110102

sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100sucB70010101010101010100

ybcF600011010101001100.10.92.9ybcF60010100110101010101ybcF60010011010101010012ybcF60010100110101010012ybcF60010011001101010102ybcF6001001010.10.9101010102.9ybcF60010011010101010101ybcF60010011010101010101ybcF60010101010011001102ybcF600101010101010100.10.90.9ybcF60010101010101010100ybcF60010101010011010101

priC50010010110101010013priC50010100101011010103priC50010011010101001013priC50010101010101010011priC50001011001101010014priC50001011001100110104priC50010011010101010101priC50010010110011010014priC50010101010101001101priC50001101001011010014priC50010101001101001013priC50010101010010110013

ybaA4000.80.20.90.10.90.10.70.3100.90.1100.70.31.1ybaA4000.30.70.60.40.50.50.70.30.60.40.60.4100.30.73.4ybaA4000.20.80.30.7100.60.410100.70.30.60.42.6ybaA40010100.40.6100.30.710100.40.61.9ybaA4000.50.50.30.7100.90.1100.40.6100.70.32.2ybaA4000.40.60.30.70.20.80.70.30.50.5010.60.40.50.54.8ybaA4000.90.10.80.20.20.80.90.110100.60.40.80.21.8ybaA4000.20.80.20.80.60.40.80.20.50.510100.80.22.9ybaA4000.70.30.50.50.50.5100.50.5100.60.4102.2ybaA4000.50.510100.60.40.50.510100.70.31.7ybaA4000.70.310100.50.50.50.510100.40.61.9ybaA40010101010100.40.6100.60.41

Lon30010100.20.810100.30.710101.5Lon3000.80.2101010101010100.2Lon3000.10.9101010101010100.9Lon30010101010101010100Lon3000.10.91010101010100.30.71.6Lon300100.20.801100.20.8010.20.8104.4Lon30010100.20.810101010100.8Lon300101010100.20.81010100.8Lon3000.20.81010100.50.5100.40.6101.9Lon3000.10.91010101010100.40.61.5Lon30010101010101001101Lon30010101010100.30.710100.7

pgpA20010101010100.20.801012.8pgpA20010101010100110012pgpA20010011001101001014pgpA20010101010101010100pgpA20010101010101010100pgpA20010100101100110103pgpA20010101001101001013pgpA20010011001011010103pgpA20001100110011010103pgpA20001101001101010102pgpA20010101001011010102pgpA20010101010010110013

yaiS15010101010101010100yaiS150011010100.50.510100.10.92.4yaiS1501010101010100.60.40.80.20.6yaiS150101010100.50.51010100.5yaiS15010101010101010100yaiS15010101010011010101yaiS15010101010101010100yaiS15010101010101010100yaiS15010101010101010100yaiS15010101010101010100yaiS1501010100.30.7101010011.7yaiS15010101010101010011

frmR10001100.60.40.60.4101010101.8frmR100011010101001100.10.92.9frmR1001001101010100.80.20.40.61.8frmR100101010100.20.81010100.8frmR10010101010101010100frmR10001010110011010104frmR10010101010101010100frmR10010101010101010100frmR10010101010101010100frmR10010101010101010100frmR1001010100.40.6011010012.6frmR10010101010101010011

kankankankan0.90.1kankankankankankankankan

7.73.39.91.18.729.31.710.90.17.441016.84.2Mut/clone7.73.310.60.47.43.69.51.58.92.1651106.84.2Mut/clone8.42.6471109.61.41101107.73.35.65.4Mut/clone1101108.52.51107.93.11101107.64.4Mut/clone6.44.67.23.81108.92.11101011108.22.8Mut/clone6.64.46.44.65.25.86.74.36.54.5566.84.210.40.6Mut/clone10.10.98.82.29.41.69.41.61101109.61.49.81.2Mut/clone8.42.65.35.76.94.19.81.26.74.311011083Mut/clone7.83.28.52.58.62.41107.53.51106.44.6110Mut/clone6.64.41101107.93.19.51.51101107.23.8Mut/clone10.60.41101106.54.55.85.2110926.24.8Mut/clone1101101101106.24.86.54.51106.54.5Mut/clone

CALLUAA311112141.7534311553.1428571429371253.63343.33333333334412032.3333333333456446404.1251322121.3753644434322252.843143055253.45443

311112143431155371253344412034564464013221236444332225431405525544

311112143431150537010025003030044412000345644640132200123644400332202050400310040005502500005404

17.320.19.914.334.48.920.916.212.816.913.8

BinFrequency

03Total # Mutations:

112

210

312

416

59

63

71

More0

BinFrequency

033

112

210

312

416

59

63

71

More0

0

0

0

0

0

0

0

0

# mutations/clone

Frequency

11 Oligo Pool Distribution (2 um/oligo)

0

0

0

0

0

0

0

0

0

Intelligent Design & Metabolic Evolution LenskiCitrate utilizationPalssonGlycerol utilizationEdwards Radiation resistanceIngramLactate productionStephanopoulosEthanol resistance MarliereThermotoleranceJ&JDiarylquinoline resistance (TB)DuPont1,3-propanediol productionChurchTrp/Tyr exchange

Cross-feeding symbiotic systems:aphids & Buchneraobligate mutualismnutritional interactions: amino acids & vitaminsestablished 200-250 million years agoclose relative of E. coli with tiny genome (618~641kb)Aphidshttp://buchnera.gsc.riken.go.jpMILKFTWVMILKFTWV HR

Pink= enzymes apparently missing in BuchernaShigenobu 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 best co-growth Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome 2005 Science SecondPassageFirst PassageSynthetic genome pair evolution

Co-evolution of mutual biosensors/biosynthesissequenced across time & within each time-pointIndependent lines of TrpD & TyrD co-culture

5 OmpF: (pore: large,hydrophilic > small) 42R-> G,L,C, 113 D->V, 117 E->A

2 Promoter: (cis-regulator) -12A->C, -35 C->A

5 Lrp: (trans-regulator) 1bD, 9bD, 8bD, IS2 insert, R->L in DBD.

Heterogeneity within each time-point . Reppas, Shendure, PoreccaAt late times Tyr- becomes prototroph!

Documents

Fabricating with DNA