Biobricks: BBa_

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Biobricks BBa_
--gca EcoRI NotI t XbaI gt SpeI a NotI PstI gct--
Standard Assembly
Parallel Rolling Assembly
Idempotent Vector Design for Standard Assembly of
Biobricks, Tom Knight 2001 http//web.mit.edu/synb
io/release/docs/biobricks.pdf http//biobricks.ai.
mit.edu/Assembly/
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Free-style FSa_

http//www.nature.com/nature/comics/syntheticbiolo
gycomic/
Hydrogen production by Cyanobacteria Dutta et
al. Microb Cell Fact. 2005
Solar-Powered Sea Slugs Rumpho et al. Plant
Physiol 2000
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Free-style FSa_
Construction Fusion PCR (Combinatorial)
(non)restriction ligation Stapling Ligation
independent cloning (LIC) Homologous
recombination (l-red, etc.) Conjugative
hierarchical assembly (no DNA preps) Post-constru
ction Selection screening Size, sequence,
function
Combinatorial polyketide biosynthesis by de novo
design and rearrangement of modular polyketide
synthase genes. Nat Biotech 2005
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How? 10 Mbp of oligos / 1000 chip
( 2 E.coli genomes or 20 Mycoplasmas /chip)
Digital Micromirror Array
1000X lower oligo costs

• 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

Amplify pools of 50mers using flanking universal
PCR primers and three paths to 10X error
correction
Tian et al. Nature. 4321050 Carr Jacobson
2004 NAR Smith Modrich 1997 PNAS
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Organism Menu
Species 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
All small ribosomal proteins
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
Western blot based on His-tags
Tian et al. Nature. 4321050
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rE.coli new in vivo genetic codes
Freeing 4 tRNAs, 7 codons UAG, UUR, AGY,
AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high
serum stability
TTT F 30362 TCT S 11495 TAT Y 21999 TGT C 7048
TTC F 22516 TCC S 11720 TAC Y 16601 TGC C 8816
TTA L 18932 TCA S 9783 TAA STOP STOP 2703 TGA STOP 1256
TTG L 18602 TCG S 12166 TAG STOP STOP 326 TGG W 20683
CTT L 15002 CCT P 9559 CAT H 17613 CGT R 28382
CTC L 15077 CCC P 7485 CAC H 13227 CGC R 29898
CTA L 5314 CCA P 11471 CAA Q 20888 CGA R 4859
CTG L 71553 CCG P 31515 CAG Q 39188 CGG R 7399
ATT I 41309 ACT T 12198 AAT N 24159 AGT S 11970
ATC I 34178 ACC T 31796 AAC N 29385 AGC S 21862
ATA 5967 ACA T 9670 AAA K 45687 AGA R 2896
ATG M 37915 ACG T 19624 AAG K 14029 AGG R 1692
GTT V 24858 GCT A 20762 GAT D 43719 GGT G 33622
GTC V 20753 GCC A 34695 GAC D 25918 GGC G 40285
GTA V 14822 GCA A 27418 GAA E 53641 GGA G 10893
GTG V 35918 GCG A 45741 GAG E 24254 GGG G 15090
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Isaacs Church Forster Carr Jacobson Jahnz Schu
ltz
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2
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4 paths to enhanced recombination
1. Homologous (e.g. l red) 2. Integrase/recombinas
e
3. ZnF-nucleases 4. Meganucleases (Baker, below)

• Design, activity, and structure of a highly
  specific artificial endonuclease. Mol Cell. 2002
  Oct10(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 Jul12(1)187-98.Aihara H, Kwon HJ,
  Nunes-Duby SE, Landy A, Ellenberger T.

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Integrase-Counter Design in BioBricks
Collins, Church, Endy, et al.
http//parts2.mit.edu/
? Xis AAV
ECFP AAV
? Int LVA
p22 attP
Reverse Terminator
p22 attB (rev comp)
l Half Bit
BBa_E0024
BBa_I11020
BBa_I11021
BBa_I11033
BBa_B0025
BBa_I11032
BBa_I11060
P22 Xis AAV
EYFP AAV
p22 Int LVA
? attP
Terminator
? attB (rev comp)
p22 Half Bit
BBa_E0034
BBa_I11030
BBa_I11031
BBa_I11023
BBa_B0013
BBa_I11022
BBa_I11061
Lewis and Hatfull, Nuc. Acid Res., 2001, Vol. 29,
2205-2216 Andersen, Applied and Environmental
Microbiology, 1998, 2240-2246
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Combinatorial Synthesis ofGenetic Networks
Guet et al. Science 2002
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