Chassis

• Parts jukebox : Meta-device : few strains

• Focus of system designs & integration

• Standardization, composability, abstraction

• Interface with environment, power supply

• Interface with computers: CAD, measurements

• Human practice: Safety & process engineering

Chassis Goals

Genome Engineering - Multiplex Automation • Accelerated evolution (GE-MASS targeting) • Decelerated evolution in production (e.g. Tn- & antimutators)• Data > model > CAD for efficient allele replacement Why genome-wide chassis engineering?• Codon changes for multi-virus resistance• Eliminate transposons (Tn-)• Change restriction sites• Orthogonal expression systems & compartments

Synergy with Thrusts & Testbeds

• Parts & Devices – chassis for surface display devices, eliminate restriction sites (CTAG); Construction Optimization Group (COG) meetings; IGEM

• Human Practices – motivation for genome isolation (via codons), ecological-testing, http://www.ia-sb.eu/

• Tumor-killing bacterium (TKB) – inherent release hence safety features – metabolic “disabling tech”

• Microbial Chemical Factory (MCF) – multi-promoter & multi-RBS tuning via GE-MASS; Tn- stability

Thrust 3: Chassis Design & Characterization 11:00-11:20 AM Tue 4-Mar-2008Carlos Bustamente, Anton Vila-Sanjurjo – mt-minigenome Drew Endy, Barry Canton – orthogonal expressionTom Knight – Mesoplasma florum Farren Isaacs, Harris Wang – MAGE & new codesDuhee Bang, Mike Jewett - in vitro & chiral engineering

Thrust 3: Chassis Design & Characterization 10:00-10:30 AM Tue 21-Sep-2008Brad Zamft, Carlos Bustamante, Natalie Kuldell– The Mitochondria as a Minimal Chassis: Expanding the Toolkit for Mitochondrial Genomic EngineeringFarren Isaacs &Harris Wang – GEMASS & new codesMike Jewett - in vitro chassis

Why Minigenomes

Tom Knight Mesoplasma florum ~750 genesCarlos Bustamante Mitochondrion ~1500 genesMike Jewett E.coli in vitro 151 genes

• Ease of synthesis• More radical designs • Existing applications -Roche Rapid Translation System (RTS) -Ambion ActivePro In Vitro Translation Kit -Novagen, Promega, Invitrogen, Qiagen Stratagene, Paragon, Amersham

113 kbp DNA 151 genes

Pure translation:Forster & Church

MSB ’05 GenomeRes.’06

Shimizu, Ueda ’01

Not minimal:High speed &

accuracy requires a few extra genes

(E.coli 20 min. doubling)

Reconstituted ribosomes:Jewett & Church

Despite early success in 1968, assembly of ribosomes under

conditions compatible with protein synthesis has been elusive

(until Aug 2008)

50S and 30S ribosomal subunits simultaneously reconstituted at

37°C and constant [Mg2+] express active luciferase

50S and 30S ribosomal subunits simultaneously reconstituted at

37°C and constant [Mg2+] express active luciferase

0

50

100

150200250

300350

Luciferase(ng/mL)

0102030405060708090

Luciferase(ng/mL)

Improving process yield & safety: trade-off

or win-win?

What do most biological systems fear?

What do all viruses have in common?

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