Microfluidics for Gene Fabrication Peter Carr & David Kong MIT Media Laboratory

Uses for DNA On-Demand base pairs single genes* genetic circuitsgenome rewrite minimal life

Recoding E.coli: rE.coli 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

SIRV-1: hjc gene parse Primers: t 1 = b 16 = 25 nt Construction oligos: b 2  t 15 = 48 nt total gene: = 390 bp 25 nt CAGGTAATTCCATATGAACATCCGTCAGTCTGGTAAATACTACGAGTACAAAACTCTGGAGATCCTGGAAAAGAATGGTTTCAAAGCGCTGCGTATCCCGGTTTCTGGTACCGGCAAACAGGCGCTGCC GTCCATTAAGGTATACTTGTAGGCAGTCAGACCATTTATGATGCTCATGTTTTGAGACCTCTAGGACCTTTTCTTACCAAAGTTTCGCGACGCATAGGGCCAAAGACCATGGCCGTTTGTCCGCGACGG GGACCTGATCGCGACCAAAAACAACACCATCTACCCTATTGAAGTTAAATCTACCTCTAAAGACGTTGTTACCGTTCGTAATTTCCAGATCGAAAAACTGTTCAAATTCTGCGCGAAATCTTCAACTTCTGTGA CCTGGACTAGCGCTGGTTTTTGTTGTGGTAGATGGGATAACTTCAATTTAGATGGAGATTTCTGCAACAATGGCAAGCATTAAAGGTCTAGCTTTTTGACAAGTTTAAGACGCTTTAGAAGTTGAAGACACTCT ATGCCACCCGCTGGTAACCGTTTACTACAAGAAATACAAAATCGTTATCGTTTATGAACTGTCTCAGGACGTTCGCACCAAAGAAAAAATCAAGTTCAAGTACGGCATCAACTCCTAACTCGAGCGGAC TACGGTGGGCGACCATTGGCAAATGATGTTCTTTATGTTTTAGCAATAGCAAATACTTGACAGAGTCCTGCAAGCGTGGTTTCTTTTTTAGTTCAAGTTCATGCCGTAGTTGAGGATTGAGCTCGCCTG t1t1 t3t3 b2b2 b4b4 t7t7 t9t9 b6b6 b 12 b 14 t5t5 t 11 t 13 t 15 b 16 b8b8 b 10 CAGGTAATTCCATATGAACATCCGTCAGTCTGGTAAATACTACGAGTACAAAACTCTGGAGATCCTGGAAAAG CAAAGCGCTGCGTATCCCGGTTTCTGGTACCGGCAAACAGGCGCTGCC CCATTAAGGTATACTTGTAGGCAGTCAGACCATTTATGATGCTCATGT CTAGGACCTTTTCTTACCAAAGTTTCGCGACGCATAGGG CGTTTGTCCGCGACGG CGACCAAAAACAACACCATCTACCCTATTGAAGTTAAATCTACCTCTA GACGTTGTTACCGTTCGTAATTTCCAGATCGAAAAACTGTTCAAATT GCGAAATCTTCAACTTCTGTGA CCTGGACTAGCGCTGGTTTTTGTTGTGGTAGA GGGATAACTTCAATTTAGATGGAGATTTCTGCAACAATGGCAAGCATT GGTCTAGCTTTTTGACAAGTTTAAGACGCTTTAGAAGTTGAAGACACT ATGCCACCCGCTGGTAACCGTTTACT CAAGAAATACAAAATCGTTATCGTTTATGAACTGTCTCAGGACGTTCG CCAAAGAAAAAATCAAGTTCAAGTACGGCATCAACTCCTAACTCGAGC GGCGACCATTGGCAAATGATGTTCTTTATGTTTTAGCAATAGCAAATACTTGACAGAGTCCTGCAAGCGTGGTTTCTTTTTTAGTTCAAGTT CGTAGTTGAGGATTGAGCTCGCCTG

SIRV-1: hjc one-step PCA t1t1 t3t3 t5t5 t7t7 t9t9 t 11 t 13 t bp b2b2 b4b4 b6b6 b8b8 b 10 b 12 b 14 b bp 108 bp 50 bp

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 Correction Protocols Hybridization-selection Mismatch Binding/RemovalMismatch Cleavage bind MutS remove MutS + mismatch (error-free DNA) Tian et al. Nature 432 (2004)Carr et al. NAR 32 (2004)Smith & Modrich PNAS 94 (1997)

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

Making it Fast & Cheap: Challenges Harnessing the full potential of oligo microarrays –minute amounts of material: amplification? –assembly: erratic behaviors of increasingly complex mixes Minimizing expensive reagents (polymerases, error correction) High throughput parallel sample handling Process integration

user designs DNA Microfluidic Gene Synthesis: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone sequence/QC express/assay DATA(MOLECULES) DATA assemble DNA constructs

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

Kong et al. Nucleic Acids Research, 2007 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

Error Type Microfluidic Device PCR tube DeletionSingle-base1916 DeletionMultiple-base55 TransitionG/C to A/T36 TransitionA/T to G/C02 TransversionG/C to C/G00 TransversionG/C to T/A11 TransversionA/T to C/G10 TransversionA/T to T/A00 Total Errors:2930 Bases Sequenced:16,25013,389 Error Rate (per base): verified identity of each gene by sequencing -12.5% of clones were error-free in agreement with theoretical predictions 1 in in in 10,00 Kong et al. Nucleic Acids Research, 2007 Carr et al. Nucleic Acids Research, 2004 Sequencing results Parallel microfluidic gene synthesis

user designs DNA Microfluidic Gene Synthesis: Integration Road Map oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone sequence/QC express/assay DATA(MOLECULES) DATA oligo microarray synthesis assemble DNA constructs

-Perform synthesis without pre-assembly amplification -Enables increased utilization of high-density DNA microarrays by: -reducing pool complexity -limiting undesired oligo interactions -maintaining reagent concentrations at desired levels Integrated Microarray-Microfluidics AgilentNimblegen (MAS) Oligo spot feature size 135  m16  m Area of spot 1.4x10 4  m x10 2  m 2 Oligo density0.1 picomole/mm 2 Maximum expected yield per spot1.4 femtomole femtomole Dimensions of spot spacing 212  m by 188  m 25 μm by 25 μm Minimal footprint of 16 oligo spots 6.4x10 5  m 2 1x10 4  m 2 Minimal chamber volume (10  m height) 6.4 nanoliters100 picoliters Estimated concentration of each oligo220 nM256 nM AgilentNimblegen (MAS) Oligo spot feature size 135  m16  m Area of spot 1.4x10 4  m x10 2  m 2 Oligo density0.1 picomole/mm 2 Maximum expected yield per spot1.4 femtomole femtomole Dimensions of spot spacing 212  m by 188  m 25 μm by 25 μm Minimal footprint of 16 oligo spots 6.4x10 5  m 2 1x10 4  m 2 Minimal chamber volume (10  m height) 6.4 nanoliters100 picoliters Estimated concentration of each oligo220 nM256 nM

user designs DNA oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone sequence/QC express/assay DATA(MOLECULES) DATA larger scale assembly assemble DNA constructs Microfluidic Gene Synthesis: Integration Road Map

Hierarchical gene synthesis

E 300 nL reactors On-chip mixing of synthesized fragments A, B, with a “rejuvenating mixture” of dNTPs, polymerase, and amplifying primers Fragment A, B, and E sample collection (real time)

user designs DNA oligo microarray synthesis assemble DNA constructs DNA error correction larger scale assembly clone sequence/QC express/assay DATA(MOLECULES) DATA express/assay assemble DNA constructs Microfluidic Gene Synthesis: Integration Road Map

Integrated gene and protein synthesis Fluorescence from GFP expressed in a PDMS microfluidic device oligos  gene  protein Fluorescence from expressed synthetic EGFP oligos  gene  protein 45 nL gene synthesis reactors, 12 nL protein synthesis reactors

Lu Chen Kelly Chang Byron Hsu Dr. Shuguang Zhang Prof. George Church Prof. Franco Cerrina Prof. Joseph Jacobson Funding: MIT Media Lab Center for Bits and Atoms (NSF) Thank You