1

Biobricks: BBa_

--gcaEcoRI NotI

tXbaI

gtSpeI

aNotI PstI gct--

Idempotent Vector Design for Standard Assembly of Biobricks, Tom Knight 2001http://web.mit.edu/synbio/release/docs/biobricks.pdfhttp://biobricks.ai.mit.edu/Assembly/

Standard AssemblyParallel

& Rolling Assembly

2

Free-style: FSa_

Hydrogen production by Cyanobacteria Dutta et al. Microb Cell Fact. 2005

Solar-Powered Sea Slugs Rumpho et al. Plant Physiol 2000

http://www.nature.com/nature/comics/syntheticbiologycomic/

3

Free-style: FSa_Construction: Fusion PCR(Combinatorial) (non)restriction ligationStapling Ligation independent cloning (LIC)Homologous recombination (-red, etc.)Conjugative hierarchical assembly (no DNA preps)

Post-construction: Selection & screening: Size, sequence, function

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nat Biotech 2005

4

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

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Organism MenuSpecies Generation

(hr)Genome size

(Mbp)Homologous recombination

Library size, comments

In vitro .01 to 24 .0002 1 1E15

phage .08 .008 1E-2 1E10

E.coli .3 5 1E-4 1E9, tame pathogen

S. cerevisiae 1.5 12 1E-3 1E7, tasty

Synechocystis 3 3.5 1E-3 ?, green

C.elegans 48 100 RNAi 1E6, can of worms

Mouse stem cells 24 3000 1E-6 & RNAi 1E6,

cute

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Combining two genomes in one cell: Stable cloning of the Synechocystis

genome in the Bacillus genome

Itaya, Fujita et al. PNAS 2005

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Yeast recombination YAC-HAC

Rapid generation of long synthetic tandem repeats and its application for analysis in human artificial chromosome formation Ebersole, et al. NAR 2005

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Genome engineering

Top Design (BioJade etc.) Detailed Design (CAD-PAM)

Synthesis (chip & error correction)

Sequence Combinatorics Evolution Sequence

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

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Full codon remapping for enhanced protein expression

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, lower %GC

Tian et al. Nature. 432:1050

20w 20m 17w 17m 16w 16m

10kd

W: wild-typeM: modified

Western blot based on His-tags

All small ribosomal proteins

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rE.coli: new in vivo genetic codes

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

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4 paths to enhanced recombination

• Design, activity, and structure of a highly specific artificial endonuclease. Mol Cell. 2002 Oct;10(4):895-905. Chevalier BS, Kortemme T, Chadsey MS, Baker D, Monnat RJ, Stoddard BL. • A conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell. 2003 Jul;12(1):187-98.Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T.

1. Homologous (e.g. red)2. Integrase/recombinase

3. ZnF-nucleases 4. Meganucleases (Baker, below)

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Integrase-Counter Design in BioBricks

λ Xis +AAV

ECFP +AAV

λ Int+ LVA

BBa_E0024 BBa_I11020 BBa_I11021

p22 attP

BBa_I11033

Reverse Terminato

rBBa_B0025

p22 attB (rev

comp)BBa_I11032 BBa_I11060 :

P22 Xis

+AAV

EYFP +AAV

p22 Int+ LVA

BBa_E0034 BBa_I11030 BBa_I11031

λ attP

BBa_I11023

Terminator

BBa_B0013

λ attB (rev

comp)BBa_I11022 BBa_I11061 :

Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29, 2205-2216Andersen, Applied and Environmental Microbiology, 1998, 2240-2246

Half Bit

p22 Half Bit

Collins, Church, Endy, et al. http://parts2.mit.edu/

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Combinatorial Synthesis ofGenetic Networks

Guet et al. Science 2002

14

.