Error-Corrected DNA Synthesis Peter Carr MIT Media Laboratory

ACTG DNA and a few handy tools ACTG Polymerase reads (copies) DNA TACTG ACTG TA ACTG TAC CAGT GGTA MutS sticks to mistakes ACTG TAG CAGT GGTA T CAGT DNA comes in complementary strands (A with T; G with C)

Uses for DNA On-Demand base pairs genetic circuitsgenome rewrite minimal life single genes* INSERT: your favorite gene(s)

Recoding E.coli: rE.coli (with Church & Isaacs) Step2. >4000 changes remove rare Arg coding free two more codons Step 3. >70,000 changes swap Leu and Ser codons orthogonal genetic codes! Step1. >300 changes TAG stops become TAA stops leaves one codon free E. Coli MG 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 designs DNA DNA Fabrication Platform: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone express/assay sequence/QC MOLECULESDATADEVICE DATA DEVICE (molecules) oligo microarray synthesis

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 >10 5 oligos per microarray >5 megabases of DNA information == Tian & Church (2004): ~600 oligos 21 genes 15 kb construct

user designs DNA DNA Fabrication Platform: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone express/assay sequence/QC MOLECULESDATADEVICE DATA DEVICE (molecules) oligo microarray synthesis assemble DNA constructs

Polymerase Construction and Amplification (PCA)

Four parallel 500-nL reactors Parallel microfluidic gene synthesis PDMS1 PDMS2 PDMS3 Fabrication

Parallel microfluidic gene synthesis Gene Total size (nt) Number of oligos Construction Oligo size (nt) Amplifying Primer sizes (nt) alba , 32 hjc , 25 ble , 45 DsRed , 20 OR , 37 GFP , 29 Kong et al. Nucleic Acids Research

user designs DNA DNA Fabrication Platform: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone express/assay sequence/QC MOLECULESDATADEVICE DATA DEVICE (molecules) DNA error correction

Error Correction for DNA ACTGACTTGCTG CAGT CAGT AAGT ACTG CAGT >10 9 copies in solution probability of correlated errors is low iteration (with strand re- partnering) makes more robust (keep) ACTG CAGT (edit) (remove)

DNA Error Removal bind MutS remove MutS + mismatch (error-free DNA) Carr et al. NAR 32 (2004) 1 error per 10 4 base pairs

user designs DNA DNA Fabrication Platform: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone express/assay sequence/QC MOLECULESDATADEVICE DATA DEVICE (molecules) larger scale assembly assemble DNA constructs

Hierarchical gene synthesis 10 3 – 10 4 base pairs

user designs DNA DNA Fabrication Platform: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone express/assay sequence/QC MOLECULESDATADEVICE DATA DEVICE (molecules) express/assay assemble DNA 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 Kong Jason Park Prof. Franco Cerrina Prof. George Church Dr. Shuguang Zhang Funding: MIT Media Lab Center 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?

DNA Error Correction Protocols Hybridization-selection Tian et al. Nature 432 (2004) Mismatch Binding/Removal bind MutS remove MutS + mismatch (error-free DNA) Carr et al. NAR 32 (2004) Mismatch Cleavage Smith & Modrich PNAS 94 (1997) 1 error per 10 4 base pairs