Multiplex DNA synthesis and some applications

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Multiplex DNA synthesis and some applications

• Farren Isaacs
• June 22, 2005
• ALife Boston
• Church Lab
• Department of Genetics
• Harvard Medical School

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Genome Sequencing Technologies the framework
gtENST00000262479 p53GCAGCCAGACTGCCTTCCGGGTCACTG
CCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAG
GAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTC
CCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATA
TTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATG
CCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGC
GGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCC
AGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCT
GGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGAT
GTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCA
CACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCA
CAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTC
AGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAA
ATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTG
GTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCA
CTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGC
CCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGA
CGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCG
CACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGC
CCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCC
CAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCG
TGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAAC
TCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCC
AGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACT
CATGTTCAAGACAGAAGGGCCTGACTCAGAC
The sequence provides the framework upon which
all the genetics, biochemistry physiology, and
ultimately phenotype depend. It provides the
boundary for scientific inquiry. The sequence is
only the first level of understanding the genome.
All genes and control elements must be
identified their functions in concert as well as
in isolation, defined their sequence variation
worldwide described and the relation between
genome variation and specific phenotypic
characteristics determined. Now we know what we
have to explain.
J.C. Venter et al. Science 291 (2001)
Shendure J, Mitra R, Varma C, Church GM, 2004
Nature Reviews of Genetics
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Sequencing Technologies
Systems Biology
Synthetic Biology
Synthesis Technologies
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Cellular Phone Designed and built by engineers
EVERY component is characterized
Cellular Network Exhibit remarkably robust,
precise behavior in the absence of our
understanding
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Synthetic Biology

• Construction of small gene networks from
  well-characterized biological parts, guided by
  models

Toggle Switch Gardner, Cantor Collins Nature
403 (2000)
Repressilator Elowitz Leibler Nature 403 (2000)
Good Review Hasty, McMillen Collins Nature
420 (2002)
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Synthetic Biology

• Design of new biological parts

Engineered Riboregulators Isaacs et al. Nature
Biotech 22 (2004)
Ligand-controlled Riboregulators Bayer Smolke
Nature Biotech 23 (2005)
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Biological Complexity
Synthetic Biology ?? Systems Biology
reduce the complexity of networks from natural
complex biological setting to isolate and study
modular components that perform a specific
function
Advanced Synthesis Technologies
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Multiplex DNA Synthesis from Programmable
Microchips
Tian et al. Nature 432 (2004)
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Cell Counter (IGEM Summer '04)

• Boston University
• Will Blake
• Jim Flanigon
• Farren Isaacs
• Ellen OShaughnessy
• Neil Patel
• Margot Schomp
• Jim Collins

• Harvard University
• John Aach
• Patrik D'haeseleer
• Gary Gao
• Jinkuk Kim
• Xiaoxia Lin
• Nathan Walsh
• George Church

http//theory.med.harvard.edu/SynBio/
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Phage Int/Xis system
Phage attachment sites attP
P
P
O
O
B
B
attB Bacterial attachment sites
Int

Xis
Int
Integrated Left attachment sites attL
Integrated Right attachment sites attR
P
B
O
P
O
B
Stably integrated prophage
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Why Integrases Excisionases?

• High fidelity site specific recombination
• Reversible excision just as reliable as
  integration
• Specific each integrase recognizes its own att
  sites, but no others
• Numerous over 300 known Tyr integrases and 30
  known Ser integrases
• Efficient very few other factors needed to
  integrate or excise
• Extensively used Phage systems
  well-characterized and used extensively in
  genetic engineering (e.g., the GATEWAY cloning
  system by Invitrogen)

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Int/Xis system with inverted att sites
Phage attachment sites attP
Bacterial attachment sites attB
0
O
O
P
P
B
B
Int

Xis
Int
Integrated Right attachment site attR
Integrated Left attachment site attL
1
P
B
P
B
O
O
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Full Cycle of Two ½-bits
State Pulse Products
0
0
1A Int2
0
1
2A Int1 Xis1 Rpt2
1
1
1B Int2 Xis2 Rpt1
1
0
2B Int1
0
0
int2
xis2
reporter1
int2
1
attR1 term attL1
xis1
reporter2
int1
2
attP2 term attB2
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Design Composite half bits in BioBricks

• Two 2kb composite parts

? Xis AAV
ECFP AAV
? Int LVA
p22 attP
Reverse Terminator
p22 attB (rev comp)
? 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
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Synthesis Testing Can Int Xis control GFP
expression?
PLlacO
PLtetO
attP
Int
GFP_AAV
attB
pBAD
Xis
pSC101
Kan
Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25,
No. 6 1203-1210
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Trouble-shooting the Int/Xis Counter

• No detectable GFP expression
• attP sterically hinders expression?
• Solution Swap positions of attB attP
• Potential problems with plasmid copy numbers
• Noise effects cross recombination b/w plasmids
• Solution Integrate a single-copy into the
  genome via ? red recombination
• Need more variants to better characterize the
  system

Solution Multiplex DNA Synthesis
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Integrating Multiplex DNA Synthesis Synthetic
Biology
Identify Desired Sequences
Implement software to design oligos for multiplex
DNA synthesis
Parallel Construction of ALL new constructs via
multiplex DNA synthesis
Integrate Constructs into E. coli genome via ?
red recombination
High throughput Screening Selection Experiments
to isolate desired behavior
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Acknowledgements
Harvard University John Aach Patrik
D'haeseleer Gary Gao Hui Gong Jinkuk Kim Xiaoxia
Lin Jingdong Tian Sasha Wait Nathan Walsh George
Church
Boston University Will Blake Jim Flanigon Ellen
OShaughnessy Margot Schomp Jim Collins
MIT Peter Carr Chris Emig Joe Jacobson
Farren Isaacs farren_at_genetics.med.harvard.edu