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Thrust 3: Chasses Design & Characterization

Technology Integration. Future testbeds. Tumor-seeking microbe. Drug-producing microbe. system requirements. Integration. Technology Base. Parts. Chasses. Existing PDC. Devices. interdependencies. Fundamental knowledge. Design. Characterization. Standards. Abstraction.

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Thrust 3: Chasses Design & Characterization

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  1. Technology Integration Future testbeds Tumor-seeking microbe Drug-producing microbe system requirements Integration Technology Base Parts Chasses Existing PDC Devices interdependencies Fundamental knowledge Design Characterization Standards Abstraction Composability Other resources Knowledge Base Thrust 3: Chasses Design & Characterization WhoDeliverables Tom Knight Simple Chasses (e.g. Mesoplasma florum) George Church Chasses, codes, & characterization Drew Endy Virtual Machine (orthogonal DNA,RNA,Protein) Adam Arkin Modeling Jay Keasling Artificial chromosomes Kris Jones Prather Engineered Metabolism impact load

  2. George Church Duhee Bang Nick Reppas Resmi Charalel Chris Brown John Aach MCB100 students Joe Jacobson Jason Park Tiffany Yu Bram Sterling Eitan Reich Chris Emig Dave Kong Collaborators on rE.coli new code Collaborators: Shuguang Zhang, Franco Cerrina, Jindong Tian, Codon Devices, Nimblegen, Agilent, Atactic/Xeotron

  3. Quant. specs/measures/milestones: 4 goals, yr 2 Goal 1 – Components to be changed • Pathway removal (for more promoters) : # paths = 2 • Cell heterogeneity (e.g., ara transporter) : variance x0.7 • Code changes : #aa, # phage & resistance level x100 • Introduce novel chemistries into cells : sup efficiency x1.5 Goal 2 – Chasses robust to change & minimal mutations • dam,dnaEQ,mutDHLMRSTY,oxyR,polAC, recAG,ssb,topB,ung,uvrD,vsr : mutation rate x0.5 • insertion elements & transposons : mutation rate x0.1

  4. Goal 3 – Additional chromosome • Isolate exogenous gene function from native chromosome : I/O transfer function for main/plasmid/BAC • Origin: Partition, Addiction : loss rate x0.3 Goal 4 – Safety controls on the chassis • Delete phage lysogens & receptors (e.g. LamB) • Delete surface toxins (LPS) : quant sepsis, innate imm. • Low conjugation (Express traS and traT) : escape rate x.1 • Add complicated or rare auxotrophies to prevent survival outside the lab (aTc-tetR, Dap) : t1/2 = 1 to 90h; escape % x0.01 • Remove all antibiotic resistance genes : MIC x0.1

  5. New in vivo genetic code: resistant to all viruses; novel amino acids Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability 4 1 Isaacs Church Forster Carr Jacobson Jahnz Schultz 3 2

  6. Hierarchical recombination-conjugation strategy 10 stages rE.coli Strategy II: “top-down” recombination l red recombination *UAG  UAA codon replacement

  7. UAG  UAA Recombinant Design • LRH: Left Region of Homology + UAA mutation (Genomic) • SIL: Safe Insertional Site Fragment + UAA mutation (Genomic) • GSC: General Selectable Cassette (e.g., kan, cat) (Synthetic) • RRH: Right Region of Homology (Genomic) *UAG  UAA codon replacement

  8. λ Red Recombination of Crossover PCR Products *UAG  UAA ~ 20 bp overlap Tm~60C Genomic-Genomic Genomic-Synthetic -C wt 1 - 5 6 - 10 11 - 14 1 2 – 12- 1516181618 1 - 5 6 - 10 11 - 15 Positive clones for relA gene: 5, 8, 9, 11

  9. Multiplexing Crossover PCRs ^ ^ • Employ Similar Approach across ~300 UAG sites • Use orthogonal sequences at crossover site (^) for selectable marker cassette • Multiplex PCR amplifications in a single, or few, reactions

  10. Release Factor 1 (RF1) Reversible Knockout • Replace endogenous copy of RF1 (prfA) with tunable version • Interfere with translation of UAG-containing genes • hemA: Glutamyl-tRNA reductase catalyzes the first step of porphyrin biosynthesis • prfA: peptide chain release factor RF1 (targets UAG & UAA) • prmC: protein-(glutamine-N5) methyltransferase that shows activity toward polypeptide chain release factors RF1 and RF2 Transcriptional & Translational Control Transcriptional Control Isaacs, et al. Nature Biotechnology 22 (2004)

  11. 3 Exponential technologies Computation & Communication (bits/sec~m$) E.coli operons Synthesis (amu/project~M$) tRNA urea B12 Analysis (kamu~base/$) telegraph tRNA Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

  12. Autocatalytic Chasses Improvements • Synthesize two parts - join - purify • Solid phase : deblock - join - wash • Yield of < 0.1% requires selection • 20%: 2 steps/mut to 2 mutations/step • 99% multiple mutations • Design & evolution for #5.

  13. rEcoded Ecoli rE.coli Projects • 113 kbp mini-genomes ribosome-display selection • 4.7 Mbp new genetic codes protein drugs • 7*7 * 4.7 Mbp mini-ecosystems • biosensors, bioenergy, high secretors, • DNA & metabolic isolation • Top Design Utility, safety & scalability • CAD-PAM • Synthesis(chip & error correction) • Combinatorics • Evolution • Sequence

  14. Why Sequencing? Evolutionary optimization, (Ribo) Sensors, metagenomics Synthesis by Sequencing Synthesis accuracy dependent on sequencing accuracy Sequencing by Synthesis: Sequencing by Extension (SbE) Sequencing by Ligation (SbL)

  15. ‘Next Generation’ Sequencing Technology Development Multi-molecule Our role ABI/APG Seq by Ligation (SbL) 454 LifeSci Paired ends, emulsion Solexa/Lynx Multiplexing & polony CGI SbL Affymetrix Software Single molecules Helicos Biosci SAB, cleavable fluors Agilent Nanopores

  16. Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes Second Passage First Passage trp/tyrA pair of genomes shows the best co-growth Reppas, Lin & Church ; Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome(2005) Science 309:1728

  17. ompF - non-specific transport channel AAAGAT CAAGAT -12 -11 -10 -9 -8 -7 -6 Can increase import & export capability simultaneously • Glu-117 → Ala (in the pore) • Charged residue known to affect pore size and selectivity • Promoter mutation at position (-12) • Makes -10 box more consensus-like

  18. Co-evolution of mutual biosensors sequenced across time & within each time-point 3 independent lines of Trp/Tyr co-culture frozen. OmpF: 42R-> G, L, C, 113 D->V, 117 E->A Promoter: -12A->C, -35 C->A Lrp: 1bp deletion, 9bp deletion, 8bp deletion, IS2 insertion, R->L in DBD. Heterogeneity within each time-point reflecting colony heterogeneity.

  19. Societal Impact Safety – clinical, accidental, threat Surveillance – consortium started with Drew Endy, Codon, Blue Heron, DNA2.0, etc. http://arep.med.harvard.edu/SBP/Church_Biohazard04c.htm

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  21. Virtual Lab Tour Site: Harvard Medical School ‘New Research Building’

  22. Computer rm2 Desks SynBERC HMS NRB room 2384000 sq ft Offices Offices Desks Computer rm 1 Equip Rm 2 Chem hood 1 Benches Equip Rm 1 Tissue Culture & PCR setup http://arep.med.harvard.edu /photo/NRB238.pdf Cold Rm 1

  23. Autoclave Kitchen SynBERC HMS NRB room 2322000 sq ft Chem hood2 Machine shop 1 Benches Cold room Polony room Desks Conf. room http://arep.med.harvard.edu /photo/NRB232.pdf

  24. SynBERC HMS Racks for instrument prototypes

  25. SynBERC HMS Polony room:sevensequencing- by-synthesis microscopes

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