1. George Church, Carlos Bustamante, Tom Knight Year Four Site Visit March 2, 2010 Chassis

2. CAD-CAM: Agilent Chip Design / Assembly OptMAGE Genetic Design via Local Search (GDLS) Measurements: In situ sequencing Morphology & behavior Selection (evolution): Cell sorting Sensor set +/- Selector sets 2 Chassis (&BioFab) Goals George Church, Carlos Bustamante, Tom Knight Microbes, organelles, in vitro, plant, animal

3. Bakal, et al. Science 316, 1753 3 145 morphological measures

4. Bakal, et al. Science 316, 1753 4 Phenoclusters Represent Functionally Related Genes.

5. 5 In situ sequencing Zhang et al Nat Gen 38:382. Goal: Go from 12-plex to >1000-plex

6. 6 From open-access Sequencer to Bio-Fab Polonator 1. Select ‘perfect part’ sequences 2. Device characterization 3. Cell sorting (FACS) Digital micromirrors Flow- cell billion beads or cells/run Photo-labile immobilization Rich Terry

7. Ligands for existing sensors : 4 sensor mechanisms 7 Vatsan Raman DNA-protein-Ligand Riboswitch/attentuation mRNA-protein-ligand

8. Support Selections for new ligands for existing sensors 54 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rhaS rpoE rpoH rpoN rpoS soxS torR trpR tyrR 12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3- methylxanthine http://pubs.acs.org/doi/abs/10.1021/ja048634j 8

9. +/- selector sets 9 SpeciesGene (+) (-) YeastURA3 -URA FOA E.coliGalK +Gal dGal E.coliTolC SDS colicinE1 HumanHPRT HAT 6-thioG AllFP FACS FACS Sri Kosuri Agilent Chip Design Refactoring, etc.

10. #2: ds-Linear x Circle 1 step 5’>3’exo Reda/E b/T Select Zhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license) #1: ds-Circle x Circle 2 step recA+ recombination Select + counterselect Link et al J. Bact 1997 (Open-access) #3: ss-90mer x ds-Circle #4: ss-Mb x ds-Circle conjugation Costantino &Court PNAS’03 Wang et al., Nature '09 MAGE Support 4 Genome engineering strategies pKO3 E.coli 10

11. Multiplex Automated Genome Engineering (MAGE) Wang et al, Nature 2009; Costantino, Court PNAS 2003 Optimized Parameters 100X: MutS 100X:  3X: Oligo half-life: 5’ phosphothio bps 2X+: Oligo length: 90mer 5X+: secondary structure 4X: Coselection: up to 8 per cycle Cycle time: 1.5 to 2.5 hrs Highly complex oligo pools for multiplexed multi-loci modifications 11 >4 billion bp of targeted genetic variation produced per day

12. 12 MAGE Instrumentation Sep 2009

13. Copyright 2010 – Boston Engineering Corporation Project: HAR002.P2 Incubation 96-well Electroporator Motion 1 Motion 2 Sample Storage Expression Reagent DNA Queue Microfiltration Plate Reagents: H2O, EtOH, Growth Media Waste Electroporator Head Unit Dimension: 4’ x 3’ x 2.5’ 13 MAGE System Upgrade Jan-May 2010 Harris Wang

14. Copyright 2010 – Boston Engineering Corporation Project: HAR002.P2 System tracks reagent levels Mid-run Cell Sampling Scheduled or On- Demand Assign recipes by channel Displays All Errors & Events, Logs to a file Start runs synchronously or asynchronously View status of all 8 growth chambers at a glance (temperature, optical density)  LabVIEW software controls user interface and all hardware.  Fully asynchronous operation  Simple Screens, No Confusing Menu Structure  All major interactions can be completed from main screen Auto-prime & purge functions for startup, cleaning, and shutdown assistance 14 MAGE Interface

15. Wang, Isaacs, et al. Nature 2009 3 MAGE days Improved growth & High production Application example #1: 23K RBS combinations per gene Metabolic Engineering Lycopene : 20 genes up, 4 down, 2 new 15

16. Aplication example#2: Multi-virus resistance Stop codons: TAG / total  X-174 5,386 b ss-DNA 0 / 9 M13 6,407 b ss-DNA 1 / 10 MS2 3,569 b ss-RNA 2 / 4 T7 39,937 b ds-DNA 6 / 60 T4168,903 b ds-DNA 19 / 277 E.coli 4,639,675 b ds-DNA 314 /1,360,152 ncbi.nlm.nih.gov/nuccore/9626372 56718463 176120924 9627425 29366675 (7 tRNAs: RITSPGL) 16

17. 64 codons 34 tRNAs 2 RFs 21 AAs 17 Sec Adapted from Forster, Church Nature MSB 2006

18. Toward multi-virus resistant rE. coli: Conjugation assembly of 314 TAG to TAA mutants (2 5 = 32 pieces to 1) RF1 KO or conditional Virus resistance & Safety tests 1-2 3-4 1-4 5-6 1-8 7-8 5-8 9-10 1-16 11-12 9-12 13-14 9-16 15-16 13-16 17-18 1-32 19-20 17-20 21-22 17-24 23-24 21-24 25-26 17-32 27-28 25-28 29-30 25-32 31-32 29-32 18 Farren Isaacs Peter Carr

19. CAD-CAM: Agilent Chip Design / Assembly OptMAGE Genetic Design via Local Search (GDLS) Measurements: In situ sequencing Morphology & behavior Selection (evolution): Cell sorting Sensor set +/- Selector sets 19 Chassis (&BioFab) Goals George Church, Carlos Bustamante, Tom Knight Microbes, organelles, in vitro, plant, animal