Biobricks: BBa_
description
Transcript of Biobricks: BBa_
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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
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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/
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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
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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
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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
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