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A C T G
DNA and a few handy tools
A C T G
Polymerase reads (copies) DNA
TA C T GA C T G T AA C T G T A C
CAGT GGTA
MutS sticks to mistakes
A C T G T A G
CAGT GGTA
T
CAGT
DNA comes in complementary strands
(A with T; G with C)
Uses for DNA On-Demand
basepairs
102 107105104103 106
genetic circuits genome rewrite
minimal lifesingle genes*IN
SERT: your fa
vorite
gene(s)
INSERT: y
our favo
rite gene(s)
Recoding E.coli: rE.coli(with Church & Isaacs)
Step2. >4000 changesremove rare Arg codingfree two more codons
Step 3. >70,000 changesswap Leu and Ser codonsorthogonal genetic codes!
Step1. >300 changesTAG stops become TAA stopsleaves one codon freeE. Coli
MG16554.6 MB
Current “price tag” >$3 million per genome
(and we want several of these)
Trends in de novo DNA synthesis
Carlson, R. (2003) The Pace and Proliferation of Biological Technologies
user designsDNA
DNA Fabrication Platform:Integration Road Map
oligomicroarraysynthesis
assembleDNA constructs
DNA errorcorrection
larger scaleassembly
clone
express/assay
sequence/QC
MOLECULESDATA DEVICE
DATADATA DEVICE (molecules)
oligomicroarraysynthesis
inkjet-printed microarrays(e.g. Agilent)
maskless array synthesizer(e.g. Nimblegen)
High Density Oligonucleotide Microarrays
a massive feedstock of DNA building blocks
>1000x reduction in oligonucleotide costs
>105 oligos permicroarray
>5 megabasesof DNA information= =
Tian & Church (2004):~600 oligos 21 genes 15 kb construct
user designsDNA
DNA Fabrication Platform:Integration Road Map
oligomicroarraysynthesis
assembleDNA constructs
DNA errorcorrection
larger scaleassembly
clone
express/assay
sequence/QC
MOLECULESDATA DEVICE
DATADATA DEVICE (molecules)
oligomicroarraysynthesis
assembleDNA constructs
Parallel microfluidic gene synthesis
GeneTotal size (nt)
Number of oligos
Construction Oligo size (nt)
Amplifying Primer sizes (nt)
alba 327 16 38 35, 32
hjc 390 16 48 25, 25
ble 461 16 47 31, 45
DsRed 733 26 50 25, 20
OR128-1 942 32 50 31, 37
GFP 993 42 42 29, 29
Kong et al. Nucleic Acids Research 35 2007
user designsDNA
DNA Fabrication Platform:Integration Road Map
oligomicroarraysynthesis
assembleDNA constructs
DNA errorcorrection
larger scaleassembly
clone
express/assay
sequence/QC
MOLECULESDATA DEVICE
DATADATA DEVICE (molecules)
DNA errorcorrection
Error Correction for DNA
A C T G
A C T T
G C T G
CAGT
CAGT
AAGT
A C T G
CAGT
>109 copies in solution
probability of correlated errors is low
iteration (with strand re-partnering) makes more robust
(keep)
A C T G
CAGT
(edit)
(remove)
DNA Error Removal
bind MutS
removeMutS +
mismatch
(error-free DNA) Carr et al. NAR 32 (2004)
1 error per 104
base pairs
user designsDNA
DNA Fabrication Platform:Integration Road Map
oligomicroarraysynthesis
assembleDNA constructs
DNA errorcorrection
larger scaleassembly
clone
express/assay
sequence/QC
MOLECULESDATA DEVICE
DATADATA DEVICE (molecules)
larger scaleassembly
assembleDNA constructs
user designsDNA
DNA Fabrication Platform:Integration Road Map
oligomicroarraysynthesis
assembleDNA constructs
DNA errorcorrection
larger scaleassembly
clone
express/assay
sequence/QC
MOLECULESDATA DEVICE
DATADATA DEVICE (molecules)
express/assay
assembleDNA constructs
Integrated gene and protein synthesis
Parallel synthesis of three genes followed by expression of fluorescent protein (dye simulation)
Synthetic EGFP observation by confocal fluorescence microscopy
Prof. Joseph Jacobson
David KongJason Park
Prof. Franco CerrinaProf. George ChurchDr. Shuguang Zhang
Funding: MIT Media LabCenter for Bits and Atoms (NSF)
Thanks to:
1 mm
Integrated gene and protein synthesis
oligos gene protein
45 nL gene synthesis reactors x3
12 nL protein synthesis reactors x3
Can we synthesize from oligos, in parallel, genes for multiple different proteins, express them, and assay their function?