1. Rapid Prototyping for Biological Design Peter Carr CBA Bits  Biology 5/1/14 This work is sponsored by the Assistant Secretary of Defense for Research & Engineering under Air Force Contract #FA8721-05-C-0002. Opinions, interpretations, recommendations and conclusions are those of the authors and are not necessarily endorsed by the United States Government.

2. CBA Bits Bio-2 Carr 5/1/14 DNA as Digital Communication receivertransmitter Bio to Bits Bits to Bio G AT T ACA SEQUENCING SYNTHESIS

3. CBA Bits Bio-3 Carr 5/1/14 The Biomolecular Prototyping Unit (BPU) Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes Build DNA Put DNA to Work Bits-Bio Converter Bits-Bio Converter User Specs Time (hrs) Fluorescence (a.u.) Did it Work? Bits-Bio-Bits Converter Bits-Bio-Bits Converter

4. CBA Bits Bio-4 Carr 5/1/14 Microfluidics Image Source: Stephen Quake “Lab-on-a-Chip” to miniaturize thousands of experiments in parallel Todd Thorsen David Kong

5. CBA Bits Bio-5 Carr 5/1/14 Re-programming Diseased Cells Self-ID and Self-Destruct DNA for classifier circuit match cell death no match no effect cancer cell normal cell miR-21miR-17-30amiR-141miR-142(3p)miR-146a hi lo 11000 Weiss, Benenson et al. (2011) Science Genetic circuit measures a cell’s molecular fingerprint match

6. CBA Bits Bio-6 Carr 5/1/14 Microfluidic Gene Assembler (MGA) Prototype Microfluidic Reactionware Inexpensive Portable Microfluidic Controller 11000 Fingerprint to match Need one of these DNA designs to make each measurement (joining >15 different DNA parts) With Weiss, Babb, Gam Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes

7. CBA Bits Bio-7 Carr 5/1/14 Advanced Decontaminants Desired Features: Specificity to target chemicals of interest Catalysis for reduced decontaminant volumes Motility to access threat within porous materials Robustness to complex environments Bio-engineered Decon Biomolecular Prototyping Unit (BPU) to enable the design of CWA-destroying enzymes Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes

8. CBA Bits Bio-8 Carr 5/1/14 High-Throughput Architecture for Rapidly Prototyping New Enzymes Measure Express Genes Quantitate both mRNA and protein production in real time

9. CBA Bits Bio-9 Carr 5/1/14 Microfluidic Measurement of Enzyme Activity Can quantify (even weak) enzyme activity in microfluidic WT 0hr 1.75hr DS6 0hr P OH O O O demeton-S OPH + 2-ethylthioethane thiol Bond broken Fluorescent product Thiol detection agent

10. CBA Bits Bio-10 Carr 5/1/14 TTT Phe TCT Ser TAT Tyr TGT Cys TTCTCCTACTGC TTA Leu TCATAAstopTGAstop TTGTCGTAGstopTGGTrp CTT Leu CCT Pro CAT His CGT Arg CTCCCCCACCGC CTACCACAA Gln CGA CTGCCGCAGCGG ATT Ile ACT Thr AAT Asn AGT Ser ATCACCAACAGC ATAACAAAA Lys AGA Arg ATGMetACGAAGAGG GTT Val GCT Ala GAT Asp GGT Gly GTCGCCGACGGC GTAGCAGAA Glu GGA GTGGCGGAGGGG Why Engineer the Genetic code? Existing code assignments are saturated Add in new chemical functions not seen in nature Better control over engineered organisms Block viral infection TTT Phe TCT Ser TAT Tyr TGT Cys TTCTCCTACTGC TTA Leu TCATAAstopTGAstop TTGTCGTAGstopTGGTrp CTT Leu CCT Pro CAT His CGT Arg CTCCCCCACCGC CTACCACAA Gln CGA CTGCCGCAGCGG ATT Ile ACT Thr AAT Asn AGT Ser ATCACCAACAGC ATAACAAAA Lys AGA Arg ATGMetACGAAGAGG GTT Val GCT Ala GAT Asp GGT Gly GTCGCCGACGGC GTAGCAGAA Glu GGA GTGGCGGAGGGG Nature’s Code 3-letter words with a choice of 4 letters = a dictionary of 64 words TTT Phe TCT Ser TAT Tyr TGT Cys TTCTCCTACTGC TTA Leu TCATAAstopTGAstop TTGTCGTAGstopTGGTrp CTT Leu CCT Pro CAT His CGT Arg CTCCCCCACCGC CTACCACAA Gln CGA CTGCCGCAGCGG ATT Ile ACT Thr AAT Asn AGT Ser ATCACCAACAGC ATAACAAAA Lys AGA Arg ATGMetACGAAGAGG GTT Val GCT Ala GAT Asp GGT Gly GTCGCCGACGGC GTAGCAGAA Glu GGA GTGGCGGAGGGG TTT Phe TCT Ser TAT Tyr TGT Cys TTCTCCTACTGC TTA Leu TCATAAstopTGAstop TTGTCGTAGstopTGGTrp CTT Leu CCT Pro CAT His CGT Arg CTCCCCCACCGC CTACCACAA Gln CGA CTGCCGCAGCGG ATT Ile ACT Thr AAT Asn AGT Ser ATCACCAACAGC ATAACAAAA Lys AGA Arg ATGMetACGAAGAGG GTT Val GCT Ala GAT Asp GGT Gly GTCGCCGACGGC GTAGCAGAA Glu GGA GTGGCGGAGGGG with Jacobson, Church, Isaacs, Wang, LaJoie, Sterling…

11. CBA Bits Bio-11 Carr 5/1/14 in silico code simulation and design Prototyping Alternate Genetic Codes Can we predict the effect of a new genetic code on a cell? Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes Test downselected codes in vitro Install new codes in vivo Modest code change (1) Extreme code design (62, 20%) 3 edits restore

12. CBA Bits Bio-12 Carr 5/1/14 Personalized Antiviral Therapies HIV-infected patient (unique) HIV virus sequence diversity Doctor chooses among many drugs and regimens Patient IS the experiment: Mileage may vary bio – bits – bio Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes Sequence patient’s HIV diversity Synthesize HIV proteins and test against antivirals Administer personalized HIV therapy

13. CBA Bits Bio-13 Carr 5/1/14 Chemical Synthesis Assemble Genes DNA Error Correction Measure Express Genes Personalized Antiviral Therapies Sequence patient’s HIV diversity Synthesize HIV PROTEASE variants, test against antivirals Administer personalized HIV therapy no drug 50 μM Shown: Atazanavir response HIV Protease ReferenceMultidrug Resistant Mutant 1 μM 50 μM no drug

14. CBA Bits Bio-14 Carr 5/1/14 Integration User Specs Oligonucleotide Synthesis Assemble Genes DNA Error Correction Measure Express Genes Parallel microfluidic gene synthesis (Kong, Carr, Jacobson) MutS error correction (Carr, Jacobson) PEC Synthesis (Chow, Jacobson) Microfluidic Gene Assembler (Kong, Thorsen, Carr) Microfluidic gene to protein to assay (Kong, Carr, Jacboson) Microfluidic Assays (Kong, Thorsen, Carr) On-Chip Central Dogma (Kong, Thorsen, Carr)

15. CBA Bits Bio-15 Carr 5/1/14 Acknowledgements MIT Lincoln Laboratory David Kong Todd Thorsen Scott Wick Kim Hamad-Schifferli Bea Yu Whitney Young Vlad Liberman Michael Sworin Ted Fedynyshyn Eric Schwoebel Sandra Deneault Darrell Ricke Anna Shcherbina Collaborators Joe Jacobson (MIT) Neil Gershenfeld (MIT) Shuguang Zhang (MIT) George Church (Harvard) Ron Weiss (MIT) Farren Isaacs (Yale) Harris Wang (Columbia) Marc LaJoie (Harvard) Bram Sterling (Harvard) Funding ASD (R&E) DARPA NSF NIH