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Rapid Prototyping for Biological Design

Rapid Prototyping for Biological Design. Peter Carr CBA Bits  Biology 5/1/14.

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Rapid Prototyping for Biological Design

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  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. A T A C A G T DNA as Digital Communication transmitter receiver Bio to Bits Bits to Bio SYNTHESIS SEQUENCING

  3. The BiomolecularPrototyping Unit (BPU) Did it Work? Bits-Bio Converter Bits-Bio-Bits Converter Chemical Synthesis DNA Error Correction Express Genes Assemble Genes User Specs Measure Put DNA to Work Build DNA Fluorescence (a.u.) Integration for rapid design-build-test Time (hrs)

  4. Microfluidics Todd Thorsen Image Source: Stephen Quake David Kong “Lab-on-a-Chip” to miniaturize thousands of experiments in parallel

  5. Re-programming Diseased CellsSelf-ID and Self-Destruct DNA for classifier circuit cancer cell normal cell no match match Weiss, Benenson et al. (2011) Science miR-142(3p) miR-17-30a miR-146a miR-141 miR-21 Genetic circuit measures a cell’s molecular fingerprint 1 1 0 0 0 cell death no effect hi hi lo lo lo match

  6. Microfluidic Gene Assembler (MGA) Prototype Microfluidic Reactionware Fingerprint to match Chemical Synthesis DNA Error Correction Express Genes Assemble Genes Measure Need one of these DNA designs to make each measurement (joining >15 different DNA parts) Inexpensive Portable Microfluidic Controller 1 1 0 0 0 With Weiss, Babb, Gam

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

  8. High-Throughput Architecture for Rapidly Prototyping New Enzymes Express Genes Measure Quantitate both mRNA and protein production in real time

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

  10. Why Engineer the Genetic code? Nature’s Code • Existing code assignments are saturated • Add in new chemical functions not seen in nature • Better control over engineered organisms • Block viral infection 3-letter words with a choice of 4 letters = a dictionary of 64 words with Jacobson, Church, Isaacs, Wang, LaJoie, Sterling…

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

  12. Personalized Antiviral Therapies Chemical Synthesis DNA Error Correction Express Genes Assemble Genes Measure Administer personalized HIV therapy Sequence patient’s HIV diversity 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 Synthesize HIV proteins and test against antivirals

  13. Personalized Antiviral Therapies Chemical Synthesis DNA Error Correction Express Genes Assemble Genes Measure Administer personalized HIV therapy Sequence patient’s HIV diversity Synthesize HIV PROTEASE variants, test against antivirals HIV Protease Reference Multidrug Resistant Mutant no drug no drug 1 μM 50 μM 1 μM 50 μM Shown: Atazanavir response

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

  15. Acknowledgements MIT Lincoln Laboratory David Kong Todd Thorsen Scott Wick Kim Hamad-Schifferli Bea Yu Whitney Young VladLiberman 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

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