George Church, Carlos Bustamante, Tom Knight

Year Four Site VisitMarch 2, 2010

Chassis

CAD-CAM: Agilent Chip Design / Assembly OptMAGE Genetic Design via Local Search (GDLS)Measurements: In situ sequencing Morphology & behaviorSelection (evolution): Cell sorting Sensor set +/- Selector sets

2

Chassis (&BioFab) Goals

George Church, Carlos Bustamante, Tom Knight

Microbes, organelles, in vitro, plant, animal

Bakal, et al. Science 316, 17533

145 morphological measures

Bakal, et al. Science 316, 1753 4

Phenoclusters Represent Functionally Related Genes.

5

In situ sequencing

Zhang et al Nat Gen 38:382.

Goal: Go from 12-plex to >1000-plex

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From open-access Sequencer to Bio-Fab

Polonator

1. Select ‘perfect part’ sequences2. Device characterization3. Cell sorting (FACS)

Digital micromirrors

Flow- cellbillion beads or

cells/run

Photo-labileimmobilization

Rich Terry

Ligands for existing sensors : 4 sensor mechanisms

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Vatsan Raman

DNA-protein-Ligand

Riboswitch/attentuation

mRNA-protein-ligand

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/ja048634j8

+/- selector sets

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Species Gene (+) (-)

Yeast URA3 -URA FOAE.coli GalK +Gal dGalE.coli TolC SDS colicinE1 Human HPRT HAT 6-thioGAll FP FACS FACS

Sri KosuriAgilent Chip DesignRefactoring, etc.

#2: ds-Linear x Circle1 step 5’>3’exo Reda/E b/TSelectZhang et al Nat.Gen 1998 Yu et al. PNAS 2000 (GeneBridges license)

#1: ds-Circle x Circle2 step recA+ recombination

Select + counterselectLink 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 strategiespKO3

E.coli

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Multiplex Automated Genome Engineering (MAGE)

Wang et al, Nature 2009; Costantino, Court PNAS 2003

Optimized Parameters

100X: MutS100X: 3X: Oligo half-life: 5’ phosphothio bps2X+: Oligo length: 90mer5X+: secondary structure4X: Coselection: up to 8 per cycleCycle time: 1.5 to 2.5 hrs

•Highly complex oligo pools for multiplexed multi-loci modifications

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>4 billion bp of targeted genetic variation produced per day

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MAGE Instrumentation Sep 2009

Copyright 2010 – Boston Engineering CorporationProject: HAR002.P2

Incubation

96-wellElectroporator 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’

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MAGE System Upgrade Jan-May 2010

Harris Wang

Copyright 2010 – Boston Engineering CorporationProject: 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

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MAGE Interface

Wang , Isaacs, et al. Nature 2009

3 MAGE days Improved growth & High production

Application example #1: 23K RBS combinations per geneMetabolic Engineering Lycopene : 20 genes up, 4 down, 2 new

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Aplication example#2: Multi-virus resistance Stop codons: TAG / total

X-174 5,386 b ss-DNA 0 / 9M13 6,407 b ss-DNA 1 / 10MS2 3,569 b ss-RNA 2 / 4T7 39,937 b ds-DNA 6 / 60T4 168,903 b ds-DNA 19 / 277E.coli 4,639,675 b ds-DNA 314 /1,360,152

ncbi.nlm.nih.gov/nuccore/9626372 56718463 176120924 9627425 29366675 (7 tRNAs: RITSPGL)

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64 codons 34 tRNAs 2 RFs21 AAs

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Sec

Adapted from Forster, ChurchNature MSB 2006

Toward multi-virus resistant rE. coli: Conjugation assembly of 314 TAG to TAA mutants (25 = 32 pieces to 1)

RF1 KO or conditional

Virus resistance &

Safety tests

1-23-4 1-4 5-6 1-87-8 5-89-10 1-1611-12 9-1213-14 9-1615-16 13-1617-18 1-3219-20 17-2021-22 17-2423-24 21-2425-26 17-3227-28 25-2829-30 25-3231-32 29-32

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Farren Isaacs

PeterCarr

CAD-CAM: Agilent Chip Design / Assembly OptMAGE Genetic Design via Local Search (GDLS)Measurements: In situ sequencing Morphology & behaviorSelection (evolution): Cell sorting Sensor set +/- Selector sets

19

Chassis (&BioFab) Goals

George Church, Carlos Bustamante, Tom Knight

Microbes, organelles, in vitro, plant, animal