Multiplex DNA synthesis and some applications
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Transcript of Multiplex DNA synthesis and some applications
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: [email protected]