Multiplex DNA synthesis and some applications
Farren Isaacs
June 22, 2005
ALife Boston
Church Lab
Department of Genetics
Harvard Medical School
Genome Sequencing Technologies: “the framework”
>ENST00000262479 [p53]
GCAGCCAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATG
GAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCAC
TGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAG
CCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAG
TCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCC
CGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCG
ATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGG
TGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCC
CATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCG
GCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCC
TCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAG
GCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCC
GCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGAC
“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
Sequencing Technologies
Systems Biology
Synthetic Biology
Synthesis Technologies
Cellular Phone: Designed and built by engineers
EVERY component is characterized
Cellular Network: Exhibit remarkably robust, precise behavior
in the absence of our understanding
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)
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)
Synthetic Biology  Systems Biology
Biological Complexity
reduce the complexity of networks
from natural complex biological
setting to isolate and study modular
components that perform a specific
function
Modular Cell Biology
Modules: composed of many types of
molecules - DNA, RNA, proteins, small
molecules - which have discrete functions
that arise from interactions among their
components
Hartwell, Hopfield, Leibler, Murray Nature 402, C46 (1999)
Arnone & Davidson Development 124, 1851 (1997)
Advanced Synthesis Technologies
Multiplex DNA Synthesis from Programmable Microchips
Tian et al. Nature 432 (2004)
1
Int
Xis
TF4
2
Int
Xis
TF3
3
Int
Xis
TF5
4
Int
Xis
TF6
1
Int
Xis
TF4
2
Int
Xis
TF3
3
Int
Xis
TF5
4
Int
Xis
TF6
Cell Counter (IGEM Summer '04)
1
Int
Xis
TF4
2
Int
Xis
TF3
3
Int
Xis
TF5
4
Int
Xis
TF6
Boston University
Harvard University
•
Will Blake
•
John Aach
•
Jim Flanigon
•
Patrik D'haeseleer
•
Farren Isaacs
•
Gary Gao
•
Ellen O’Shaughnessy
•
Jinkuk Kim
•
Neil Patel
•
Xiaoxia Lin
•
Margot Schomp
•
Nathan Walsh
•
Jim Collins
•
George Church
http://theory.med.harvard.edu/SynBio/
Phage Int/Xis system
Phage attachment sites
attP
P O P’
B O B’
attB
Bacterial attachment sites
Int
Int
Integrated Left attachment sites
attL
B O P’
+
Xis
Integrated Right attachment sites
attR
P O B’
Stably integrated prophage
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)
Int/Xis system with inverted att sites
Phage attachment sites
attP
P O P’
Bacterial attachment sites
0
Int
Integrated Right attachment site
attR
P O B’
attB*
B’ O B
Int
1
+
Xis
Integrated Left attachment site
attL*
P’ O B
Full Cycle of Two ½-bits
State
Pulse
Products
0
Int2
Xis2
Rpt1
int
int22
xis2
rpt1
int2
xis2 reporter1
0
1A
Int2
0
1
attR
attP
attL
attB
**
attR11––term–– attL
11*1
term
1
1
Int1 Xis1
Rpt2
1
1B
Int2 Xis2
Rpt1
2B
Int1
1
0
0
0
2
Int1
Xis1
Rpt2
int1
xis1
rpt2
int1
xis1 reporter2
attP
attP
attR
–
–attB
attB
attL222***
22 –term–
2–term–
term
2A
Design Composite half bits in BioBricks
Two 2kb composite parts:
λ Half Bit
BBa_I11060 :
p22 Half Bit
BBa_I11061 :
λ Int+
LVA
p22 attP
Reverse
Terminator
p22 attB
(rev comp)
BBa_I11020
BBa_I11033
BBa_B0025
BBa_I11032
p22 Int+
LVA
λ attP
Terminator
BBa_I11030
BBa_I11023
BBa_B0013
λ attB
(rev comp)
BBa_I11022
λ Xis
+AAV
ECFP
+AAV
BBa_I11021 BBa_E0024
P22 Xis
+AAV
EYFP
+AAV
BBa_I11031 BBa_E0034
Synthesis & Testing: Can Int + Xis control GFP expression?
PLlacO
PLtetO
PLlacO PLtetO
attP
attP
Int
GFP_AAV
GFP-aav
Integrase
attB*
attB
4000
pBAD
pBAD
Test Construct1000
2
4594 bps
3000
pSC101
ColE1
Excisionase
Xis
2000
KanKan
Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25, No. 6 1203-1210
Trouble-shooting the Int/Xis Counter
PLlacO PLtetO
attP
GFP-aav
Integrase
attB
• No detectable GFP expression
• attP sterically hinders expression?
• Solution: Swap positions of attB & attP
4000
Test Construct1000
2
pBAD
4594 bps
3000
ColE1
Excisionase
2000
• Potential problems with plasmid copy numbers
• Noise effects & cross recombination b/w plasmids
•Solution: Integrate a single-copy into the genome via λ red recombination
Kan
• Need more variants to better characterize the system
RBSI
TagI
RBSX
TagX
S/FP ‘Read-out’
I-X Pairs
x2
x3
x2
x3
x2
x5
HUGE Increase in Complexity
* = variable region
360 New Test Constructs
Solution: Multiplex DNA Synthesis
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 λ red recombination
High throughput Screening & Selection Experiments to isolate desired behavior
Acknowledgements
Harvard University
John Aach
Patrik D'haeseleer
Boston University
Gary Gao
Will Blake
Hui Gong
Jim Flanigon
Jinkuk Kim
Ellen O’Shaughnessy
Xiaoxia Lin
Margot Schomp
Jingdong Tian
Jim Collins
Sasha Wait
Nathan Walsh
George Church
Farren Isaacs: farren@genetics.med.harvard.edu
MIT
Peter Carr
Chris Emig
Joe Jacobson