Multiplex DNA synthesis and some applications

Farren Isaacs

June 22, 2005ALife Boston

Church LabDepartment of GeneticsHarvard Medical School

“The sequence provides the framework upon which all the genetics, biochemistryphysiology, and ultimately phenotype depend. It provides the boundary for scientificinquiry. The sequence is only the first level of understanding the genome. All genesand control elements must be identified; their functions in concert as well as inisolation, defined; their sequence variation worldwide described; and the relationbetween genome variation and specific phenotypic characteristics determined.Now we know what we have to explain.” J.C. Venter et al. Science 291 (2001)

>ENST00000262479 [p53]GCAGCCAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCGCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGAC

Genome Sequencing Technologies: “the framework”

Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics

Systems Biology Synthetic Biology

Synthesis Technologies

Sequencing Technologies

Cellular Network: Exhibit remarkably robust, precise behavior in the absence of our understanding

Cellular Phone: Designed and built by engineers EVERY component is characterized

Synthetic Biology

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

Toggle SwitchGardner, Cantor & Collins Nature 403 (2000)

RepressilatorElowitz & Leibler Nature 403 (2000)

Good Review: Hasty, McMillen & Collins Nature 420 (2002)

Synthetic Biology

• Design of new biological parts

Engineered RiboregulatorsIsaacs et al. Nature Biotech 22 (2004)

Ligand-controlled RiboregulatorsBayer & Smolke Nature Biotech 23 (2005)

Biological Complexityreduce 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)

Synthetic Biology Systems Biology

Advanced Synthesis Technologies

Multiplex DNA Synthesis from Programmable Microchips

Tian et al. Nature 432 (2004)

Xis TF4

Xis TF3

Int

Int

Xis TF5

Xis TF6

Int

Int

1

2

3

4

Xis TF4

Xis TF3

Int

Int

Xis TF5

Xis TF6

Int

Int

1

2

3

4

Cell Counter (IGEM Summer '04)

Boston University

• Will Blake

• Jim Flanigon

• Farren Isaacs

• Ellen O’Shaughnessy

• 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/

Xis TF4

Xis TF3

Int

Int

Xis TF5

Xis TF6

Int

Int

1

2

3

4

Phage attachment sites

attP

Phage Int/Xis system

Int Int Xis+

attB Bacterial attachment sites

Integrated Left attachment sites

attLIntegrated Right attachment sites

attR

Stably integrated prophage

P’P O

B’B O

P’B O P O B’

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

Int Int Xis

Phage attachment sites

attPBacterial attachment sites

attB*

+

P’P B’ BO O0

1Integrated Right attachment site

attRIntegrated Left attachment site

attL*P BP’B’O O

Full Cycle of Two ½-bits

State

Pulse

Products

0

0

1A Int2

0

1

2AInt1 Xis1

Rpt2

1

1

1BInt2 Xis2

Rpt1

10

2B Int1

0

0

1 xis2 reporter1int2

2 xis1 reporter2int1

attR1 –term– attL1*

attP2 –term– attB2*

int2

Int2

int1

Int1

xis1

Xis1

rpt2

Rpt2

int2

Int2

xis2

Xis2

rpt1

Rpt1

int1

Int1

xis1 reporter2int1

attR2 – – attL2*

term

xis1 reporter2int1

attP2 –term– attB2*

attP1 – – attB1*

xis2 reporter1int2

term

xis2 reporter1int2

attR1–term– attL1*

Design Composite half bits 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 :

Two 2kb composite parts:

λ Half Bit

p22 Half Bit

Synthesis & Testing: Can Int + Xis control GFP expression?

Lutz and Bujard, Nuc. Acids Res., 1997, Vol. 25, No. 6 1203-1210

Test Construct 2

4594 bps

1000

2000

3000

4000

PLlacO

attP

GFP-aav

attB

ColE1

Kan

Excisionase

pBAD

Integrase

PLtetO

Xis

pBAD

Int

PLlacO PLtetO

GFP_AAV

attP

attB*

pSC101

Kan

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 λ red recombination

• Need more variants to better characterize the system* = variable region

RBSI x2TagI x3RBSX x2TagX x3S/FP ‘Read-out’ x2I-X Pairs x5

HUGE Increase in Complexity360 New Test Constructs

Solution: Multiplex DNA Synthesis

Test Construct 2

4594 bps

1000

2000

3000

4000

PLlacO

attP

GFP-aav

attB

ColE1

Kan

Excisionase

pBAD

Integrase

PLtetO

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

High throughput Screening & Selection Experiments to isolate desired behavior

Integrate Constructs into E. coli genome via λ red recombination

Acknowledgements

Boston University

Will Blake

Jim Flanigon

Ellen O’Shaughnessy

Margot Schomp

Jim Collins

Harvard University

John Aach

Patrik D'haeseleer

Gary Gao

Hui Gong

Jinkuk Kim

Xiaoxia Lin

Jingdong Tian

Sasha Wait

Nathan Walsh

George Church

MIT

Peter Carr

Chris Emig

Joe Jacobson

Farren Isaacs: farren@genetics.med.harvard.edu