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Self-Replicating Machine

C reating a “Synthetic” Bacterial Cell John Glass The J. Craig Venter Institute, Rockville, MD and San Diego, CA. Self-Replicating Machine.

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Self-Replicating Machine

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  1. Creating a “Synthetic” Bacterial Cell John GlassThe J. Craig Venter Institute, Rockville, MD and San Diego, CA

  2. Self-Replicating Machine NASA Conference Publication 2255 (1982), based on the Advanced Automation for Space Missions NASA/ASEE summer study Held at the University of Santa Clara in Santa Clara, California, from June 23-August 29, 1980

  3. Self-Replicating Machine

  4. For our purposes, we define a synthetic cell as one that operates off of a chemically synthesized genome

  5. A computer analogy -- the genome of a cell is the operating system & the cytoplasm is the hardware • The cytoplasm contains all of parts (proteins, ribosomes, etc.) necessary to express the information in the genome. • The genome contains all information necessary to produce the cytoplasm and cell envelope and to replicate itself. • Each is valueless without the other. The cytoplasm is the hardware that runs the operating system. The chromosome is the operating system.

  6. Approach used to synthesize a bacterial cell Assemble overlapping synthetic DNA oligonucleotides(~60 mers) Recipient cell Synthetic cell Cassettes (~1 kb) Assemble cassettes by homologous recombination Genome Transplantation Completely assembled synthetic genome Genome Synthesis

  7. Science August 2007 Mycoplasma mycoides Mycoplasma capricolum RECIPIENT CELLS gDNA DONOR

  8. Science February 2008 42 43 44 45 6kb 24kb 72kb 144kb 580kb yeast Yeast Vector 50-77B 50-77A Whole 1/25 1/8 1/4 Chemical Synthesis E. coli yeast

  9. Science August 2009 Yeast Mycoplasma capricolum Mycoplasma mycoides RECIPIENT CELLS gDNA DONOR

  10. Science May 2010

  11. Synthetic Organism Designer 1.0 Design Codon Opt. Oligo Synthesis

  12. Cells are complex machines with thousands of moving parts Tomography Mycoplasma pneumoniae from Proteome organization in a genome-reduced bacterium. Kuhner et al. 2009 Science 326: 1235-40

  13. Cells are 25-50% Dark Matter Small Peptides of unknown function Hypothetical Proteins Small RNAs of unknown function Epigenetic Modifications ? Moonlighting Proteins Tomography

  14. What do we mean by “minimal bacterial cell”? We consider a bacterial cell to be minimal if it contains only the genesthat are necessary and sufficientto ensure continuous growth under ideal laboratory conditions.

  15. Why make a minimal cell • To define a minimal set of genetic functions essential for life under ideal laboratory conditions. • To discover the set of genes of currently unknown function that are essential and to determine their functions. • To have a simple system for whole cell modeling. • To modularize the genes for each process in the cell (translation, replication, energy production, etc.) and to design a cell from those modules. • To build more complex cells by adding new functional modules.

  16. What bacterial cell will we minimize? • We chose to minimize Mycoplasma mycoides JCVI-syn1.0 the synthetic version of Mycoplasma mycoides because: • It has a small genome (1.08 MB). • It can be readily grown in the laboratory. • We can routinely chemically synthesize its genome and clone it in yeast as a YCp. • We can isolate the synthetic genome out yeast as naked DNA and bring it to life by transplanting it into a recipient mycoplasma cell. • We have developed a suite of tools to genetically engineer its genome.

  17. Synthesis of the Mycoplasma mycoidesJCVI-syn1.0 Genome Gibson et al., 2010 Science 329, 52-56

  18. Our starting point for minimization is the synthetic genome M. mycoides JCVI-syn1.0 There are 2 ways to minimize TOP DOWN: Start with the full size viable M. mycoides JCVI syn1.0 synthetic genome. Remove genes and clusters of genes one (or a few) at a time. At each step re-test for viability. Only proceed to the next step if the preceding construction is viable and the doubling time is approximately normal. BOTTOM UP: Make our best guess as to the genetic and functional composition of a minimal genome and then synthesize it. Craig Venter calls this the Hail Mary genome.

  19. For both approaches, we need to identify genes that are non-essential and are therefore candidates for removal. We are doing this in three ways. • Identify genes with functions that are usually non-essential such as IS elements, R-M systems, integrative and conjugative elements, etc. • Performglobal transposon mutagenesis to identify individual genes that can be disrupted without loss of viability.

  20. Tn5- puromycin global mutagenesis of M. mycoides • Illumina sequencing yielded 10,902 unique insertion sites • 754 genes hit, 160 not hit. So many genes are hit because there is extensive functional redundancy. For example, there are 2 rRNA operons and only one is necessary.

  21. Top down approach: Stepwise genome reduction

  22. Top down approach: Stepwise genome reduction M. mycoides wild type 1089 kb M. mycoides JCVI-syn 1.0 1079 kb M. mycoides JCVI-syn 1.0 – 6RM(12 genes, 17 kb) 1062 kb M. mycoides JCVI-syn 1.0 – 6RM(12 genes, 17 kb) – 6 IS (12 genes, 9 kb) 1051 kb M. mycoides JC syn1.0 – 6RM(12 genes, 17 kb) – 6 IS (12 genes, 9 kb) – ICE (44 genes, 71 kb) 980 kb M. mycoides JC syn1.0 – 6RM(12 genes, 17 kb) 928 kb – 6 IS (12 genes, 9 kb) – ICE (44 genes, 71 kb) – D5 deletions (52 kb) We plan to continue removing the large clusters, testing for viability at each step. After that, small clusters and individual non-essential genes will be removed to arrive at the minimal genome.

  23. The bottom up approach Design and synthesis of a “Hail Mary” genome Use the Tn5 transposon single gene disruption by insertion map data and our knowledge of essential functions in the cell to make the best guess as to which genes to include in a minimal genome.

  24. “Hail Mary Deletions” mapped onto M. mycoides JCVI-syn1.0 West Coast Design

  25. “Hail Mary” Minimal Genome Construction 1/8th molecules can be individually tested for functionality and mixed with subsequent designs 16,000 oligos (70 bases) ↓ 370 stage-1 assemblies (1.4 kb) ↓ 74 stage-2 assemblies (6.7 kb) ↓ 8 stage-3 assembles (50-75 kb) ↓ 483 kb Minimal Genome

  26. Hail Mary Genes by functional category KEEP DELETE Amino acid biosynthesis 0 4 Biosynthesis of cofactors 9 2 Cell envelope 28 92 Cellular processes 3 8 Central intermediary metabolism 7 8 DNA metabolism 32 32 Energy metabolism 28 35 Fatty acid and phospholipid metabolism 7 6 Hypothetical proteins 59 110 Mobile and extrachromosomal element fcns 0 14 NULL (tRNAs, rRNAs, RNAs) 49 0 Protein fate 22 23 Protein synthesis 107 8 Purines 19 7 Regulatory functions 9 8 Signal transduction 3 14 Transcription 14 4 Transport and binding proteins 35 33 Unknown function 21 47 Yeast vector and markers 4 0 ___________________________________________________________ TOTAL 457 455

  27. Design of a modular genome Can genes within individual functional categories be clustered into modules?

  28. Can all 30 tRNA genes of M. mycoides be organized in one module? 2 9 M. mycoides JCVI-syn 1.0 4 2 5 tRNA gene clusters are enlarged to show the direction of transcription. JCVI-syn 1.0 has 8 single tRNA genes and 5 clusters of 2 to 9 genes, for a total of 30. promoter tRNA gene terminator Key

  29. Moving life into the digital world and back Our capacity to build microbes capable of solving human problems is limited only by our imagination

  30. Possible future uses of synthetic & engineered species • Increase basic understanding of life • Increase the predictability of synthetic biological circuits • Become a major source of energy • Replace the petrol-chemical industry • Enhance bioremediation • Materials science – expand biology’s use of the periodic table • Drive antibiotic and vaccine discovery & production • Gene therapy via stem cell engineering

  31. It Takes a Village to Create a Cell • Algire, Mikkel • Alperovich, Nina • Assad-Garcia, Nacyra • Baden-Tillson, Holly • Benders, Gwyn • Chuang, Ray-Yuan • Dai, Jianli • Denisova, Evgeniya • Galande, Amit • Gibson, Daniel • Glass, John • Hutchison, Clyde • Iyer, Prabha • Jiga, Adriana • Krishnakumar, Radha • Lartigue, Carole • Ma, Li • Merryman, Chuck • Montague, Michael • Moodie, Monzia • Moy, Jan • Noskov, Vladimir • Pfannkoch, Cindi • Phang, Quan • Qi, Zhi-Qing • Ramon, Adi • Saran, Dayal • Smith, Ham • Tagwerker, Christian • Thomas, David • Tran, Catherine • Vashee, Sanjay • Venter, J. Craig • Young, Lee • Zaveri, Jayshree • Johnson, Justin • Brownley, Anushka • Parmar, Prashanth • Pieper, Rembert • Stockwell, Tim • Sutton, Granger • Viswanathan, Lakshmi • Yooseph, Shibu Funding fromDARPA Living Foundries Synthetic Genomics Inc. DOE GTL program

  32. DNA synthesis is getting easier, faster, and cheaper $0.15 / bp Dollars per basepair Agilent Year

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