Construction of a Bacterial Cell Containing Only Essential Genes Necessary to Impart Life
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Transcript of Construction of a Bacterial Cell Containing Only Essential Genes Necessary to Impart Life
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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
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Rockville, MD and San Diego, CA,USA
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• 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
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Number of genes in each class
n 438
e 241
i127
ie 48
in 58
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Synthesis of a Reduced Genome Design
~50% reduction in genome size
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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
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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
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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
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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
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Microfluidics for genome transplantation
James Pelletier, Elizabeth Strychalski, Nacyra Assad-Garcia, Vanya Paralanov, Andreas Mershin, Neil
Gershenfeld, John Glass
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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
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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
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step 3: condense donor genomes and cluster recipient cells
10 µm
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step 4: compress genomes and cells
10 µm
2 µm
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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
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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!