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

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 TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG326TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R 28382 CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S 11970 ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G 33622 GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG15090 Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability Isaacs Church Forster Carr Jacobson Jahnz Schultz 1 2 3 4

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

7. UAG  UAA Recombinant Design *UAG  UAA codon replacement 1.LRH: Left Region of Homology + UAA mutation(Genomic) 2.SIL: Safe Insertional Site Fragment + UAA mutation(Genomic) 3.GSC: General Selectable Cassette (e.g., kan, cat)(Synthetic) 4.RRH: Right Region of Homology(Genomic)

8. λ Red Recombination of Crossover PCR Products 1 - 5 6 - 10 11 - 14 1 - 5 6 - 10 11 - 151 2 – 12- 1516181618 -C wt Genomic-Genomic Genomic-Synthetic Positive clones for relA gene: 5, 8, 9, 11 *UAG  UAA ~ 20 bp overlap Tm~60C

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 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 Replace endogenous copy of RF1 (prfA) with tunable version –Interfere with translation of UAG-containing genes Transcriptional Control Transcriptional & Translational Control Isaacs, et al. Nature Biotechnology 22 (2004)

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

12. Autocatalytic Chasses Improvements 1.Synthesize two parts - join - purify 2.Solid phase : deblock - join - wash 3.Yield of < 0.1% requires selection 4.20%: 2 steps/mut to 2 mutations/step 5.99% multiple mutations 6.Design & evolution for #5.

13. 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 rEcoded Ecoli

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

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.  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 Second Passage First Passage Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes

17. 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 -12 -11 -10 -9 -8 -7 -6 A AAGAT C AAGAT Can increase import & export capability simultaneously ompF - non-specific transport channel

18. 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. Co-evolution of mutual biosensors sequenced across time & within each time-point

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. SynBERC HMS NRB room 238 4000 sq ft http://arep.med.harvard.edu /photo/NRB238.pdf Offices Cold Rm 1 Desks Equip Rm 1 Equip Rm 2 Tissue Culture & PCR setup Computer rm 1 Computer rm2 Benches Chem hood 1

23. SynBERC HMS NRB room 232 2000 sq ft http://arep.med.harvard.edu /photo/NRB232.pdf Polony room Cold room Machine shop 1 Conf. room Benches Chem hood2 Desks Autoclave Kitchen

24. SynBERC HMS Racks for instrument prototypes

25. SynBERC HMS Polony room: seven sequencing- by-synthesis microscopes