Learning the Rules of Genome Design

John Glass

for members of the Venter Institute Synthetic Genomics Group

The J. Craig Venter Institute, Rockville, MD and San Diego, CA

Rockville, MD and San Diego, CA,USA

• To discover the genetic kernel of life• To provide a platform for systems biologists• To learn essential design features for genomes• To modularize genomes for easier design

Why are we building a minimal cell?

Biosynthesis of cofactors, prosthetic groups, and carriersCell envelopeCellular processesCentral intermediary metabolismDNA metabolismEnergy metabolismFatty acid and phospholipid metabolismHypothetical proteinsNoncoding RNA feature

Protein fateProtein synthesisPurines, pyrimidines, nucleosides, and nucleotidesRegulatory functionsSignal transductionTranscriptionTransport and binding proteinsUnclassifiedUnknown function

Reduced, Reorganized

Number of genes in each class

n 438

e 241

i127

ie 48

in 58

Synthesis of a Reduced Genome Design

~50% reduction in genome size

Construction of 1/8 RGD + 7/8 wild type genome by Recombinase-Mediated Cassette Exchange (RMCE).

3’ URA3 MET14

1

3’ URA3 MET14

1/8 HMG5' URA3

5' URA3 3’ URA3 MET14

Cre recombinaseProm

1/8th RGD Donor PlasmidCre recombinaseProm

+

2

3

RMCE

Landing pad

Syn1 Genome

Modularization (defragmentation)

Before After

Biosynthesis of cofactors & prosthetic groups Cell envelopeCellular processesCentral intermediary metabolismDNA metabolismEnergy metabolismFatty acid and phospholipid metabolismHypothetical proteinsNoncoding RNA feature

Protein fateProtein synthesisPurines, pyrimidines, nuc’sides & nuc’tidesRegulatory functionsSignal transductionTranscriptionTransport and binding proteinsUnclassifiedUnknown function

Assemble cassettes by homologous recombination

Assemble overlapping synthetic oligonucleotides (~60 mers)

Completely assembled synthetic genome

3 Technologies Invented to Produce Synthetic Bacterial Cells

Cassettes (5-7 kb)

Recipient cell Synthetic cell

GenomeTransplantation

Genome Synthesis

Yeast Clone

It Takes a Village to Create a Cell

Support fromDARPA Living FoundriesSynthetic Genomics, Inc.

Synthetic Genomics, Inc.• Gibson, Dan

JCVI• Assad-Garcia, Nacyra • Chuang, Ray-Yuan• Gibson, Daniel • Glass, John• Hutchison, Clyde• Karas, Bogumil• Ma, Li• Merryman, Chuck

• Montague, Michael • Noskov, Vladimir• Smith, Ham • Sun, Lijie• Suzuki, Yo• Venter, Craig • Wise , Kim• Yee, Tony

• Venter, Craig

Microfluidics for genome transplantation

James Pelletier, Elizabeth Strychalski, Nacyra Assad-Garcia, Vanya Paralanov, Andreas Mershin, Neil

Gershenfeld, John Glass

How can we transfer megabases of DNA into bacteria?

Lartigue et al. Science 2009

recipient cellsMycoplasma capricolum5

µm

Whole genomes are as big as cells!

donor genomesMycoplasma mycoides

5 µm

mix donor genomes

and recipient cells

http://www.partnaranimalhealth.com/osCommerce/images/DCE-0016S%20Centrifuge%20Tube.jpghttp://ecx.images-amazon.com/images/I/215d1cMFryL._SX342_.jpg

Microfluidics for genome transplantation

high cell/DNA densities

http://www.avena-medica.com/ProductVault/product_1351077166__mg_3651_S4.jpg

10 µm

isolate donor DNA

recover cells

bulk microfluidic

reveal mechanism

precisegentlecontrol

step 3: condense donor genomes and cluster recipient cells

10 µm

step 4: compress genomes and cells

10 µm

2 µm

Microfluidics to complement genome transplantation

yeast nuclei

Gram-negativeH. influenzae

Gram-positiveS. thermophilus

next steps

• precise, gentle control• real-time visualization• high cell/genome densities• multi-parameter optimizations

Thank you very much!

John GlassNacyra-Assad GarciaVanya ParalanovEvgeniya DenisovaDavid BrownAdriana Jiga

Andreas MershinNeil GershenfeldWill LangfordPrashant PatilCharles FracchiaFei ChenPaul TillbergDavid Feldman

Elizabeth StrychalskiJason KraljJavier Atencia

You!