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Janet Nguyen Andrei Anghel Nagham Chaban Synthetic Life & Implications for Applied Microbiology What is Synthetic Biology? Definition: Synthetic biology is the engineering of biology; the synthesis of complex, biological based system, which display function that does not exist in nature. In essence, synthetic biology enables the design of biological system in rational and systemic way Drafted by the NEST High Level Expert Group Overview of Presentation J. Craig Venter Institute: • Synthesized a novel 1.1 mbp genome • Transplanted a synthetic genome into host cells and completely replaced the host genome • New cells were capable of self-replication and expressed only novel genes Overview of Presentation Topics to be Covered: 1. Genome Synthesis 2. Intercellular Transplant 3. Potential Uses of Technology Timeline of Advancements Experimental Organisms • Organisms were specifically chosen for: Size of genome Stability of genome in host Speed of replication Lack of cell wall Experimental Organisms • Donor: Mycoplasma mycoides Subspecies: mycoides Strain: Large Colony GM12 Replicates every 80 min • Recipient: Mycoplasma capricolum Subspecies: capricolum Strain: California Kid (CK) Replicates every 100 min Experimental Organisms • Mycoplasma genus 1. Genome Synthesis Janet Nguyen Synthesis: Designing the genome M. mycoides JCVI-syn1.0 Biologically significant differences were corrected Synthetic and wild type polymorphic at 19 sites Watermark sequences Sequences encode unique identifiers Limits their translation into peptides Synthesis: Interesting watermarks I. A code to interpret the rest of the watermarks and website address. II. To live to err, to fall, to triumph, to recreate life out of life. III. See things not as they are, but as they might be. IV. What I cannot build, cannot understand. Synthesis: The Genome Mycoplasma mycoides JCVI-syn1.0 Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 3. 100 kb fragments 4. Complete genome Synthesis: Strategy Hierarchical strategy: 3 Stages 1 kb → 10 kb → 100 kb → genome (1000 kb) Start with 1 kb fragments (n=1078) with 80 bp overlaps to join to neighbours chemically synthesized by Blue Heron have restriction enzyme sites at termini Synthesis: Stage 1 = 1 kb to 10 kb 1-kb fragments and a vector recombined in vivo in yeast Very active recombination system! Plasmid then transferred to E.coli Synthesis: Stage 1 = 1 kb to 10 kb 1-kb fragments and a vector recombined in vivo in yeast Very active recombination system! Plasmid then transferred to E.coli Synthesis: Stage 1 = 1 kb to 10 kb Recombinant plasmid isolated from E.coli clones All first-stage assemblies sequenced Plasmids digested to find cells with assembled 10 kb insert 19/111 had errors End of Stage 1: results in 10-kb fragments (n=109) Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 3. 100 kb fragments 4. Complete genome Synthesis: Stage 2 = 10 kb to 100 kb 10 kb fragments and cloning vectors transformed into yeast 100 kb assemblies not stably maintained in E.coli Recombined plasmid extracted from yeast Multiplex PCR presence of a PCR product would suggest an assembled 100 kb PCR products run on agarose gel End of Stage 2: Results in 100 kb fragments (n=11) Synthesis Overview 1. 1 kb fragments 2. 10 kb fragments 3. 100 kb fragments 4. Complete genome Synthesis: Complete genome assembly Isolated small quantities of each 100 kb fragment Purification: exonuclease then anion-exchange column Small fraction of total plasmid DNA (1/100) was digested Then analyzed by gel electrophoresis Result: 1ug of each assembly per 400ml of yeast culture Not all yeast chromosomal DNA removed Synthesis: Complete genome assembly To further enrich for the 100 kb fragments: Trapped plasmids digested, releases inserts Sample of each fragment mixed with molten agarose As agarose solidifies, fibers thread and “trap” circular plasmids gel electrophoresis transformed into yeast, no vector sequence required Complete genome assembled in vivo in yeast, and grown as yeast artificial chromosome Synthesis complete! Next steps: • Transplantation of genome • Verification of genome 2. Intercellular Transplantation Andrei Anghel Transplant Overview Dr. Carole Lartigue Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome Transplantation: Hurdles Genetic modifications made to DNA template in order to allow cloning entire chromosome as a plasmid in yeast Inactivation of recipient cell restriction enzyme Methylation of the synthetic genome Transplantation: Procedure • Starved M. capricolum cells were mixed with isolated, synthetic DNA • Incubated for 3 hours at 37°C to allow recovery, then plated until large blue colonies formed • Blue colonies were then used to inoculate selective broth tubes The Complete Synthetic Cell The Complete Synthetic Cell Transplantation: Verification and Efficiency • Ensuring no false-positive results was crucial • M. mycoides JCVI-syn1.0 was transformed with a vector containing a selectable tetracycline-resistance marker and a b-galactosidase gene for screening • PCR experiments and Southern blot analysis of isolated putative transplanted cells • Multiple specific antibody reactions were carried out to test for species specific proteins Transplantation: Verification Transplantation: Verification and Efficiency • Only 1 out of 48 yeast colonies contained a full genome • Only 1 in 150,000 successful transplants in the most efficient experiments • Transplant yield was optimal 5×107 cells used • Yields began to plateau at donor DNA concentrations with 107 high 3. Potential Uses of the Technology Nagham Chaban Uses of the Technology • DNA is the software of life • How could synthetic biology and DNA transfer affect our lives? • Creating synthetic bacteria and transferring man-made DNA allowed the new bacteria to live and replicate • That was proof of principle that life can be created from a computer Uses of the Technology • Designing synthetic bacteria ensures that synthetic DNA can be used for valuable things in our lives • The key is to understand how to change this software in order to create synthetic life • Can lead to powerful technology and many applications and products: biofuel, medicines, food, etc. Applications: Medicine MALARIA • Kills many people • Numerous malaria pathogens are resistant to the first generation drug • Artemisinin is a second generation drug that can treat malaria • But there is always a problem! Applications: Medicine • Artemisinin is available in low quantity in nature • Synthetic biology can be the solution by building up a new biosynthetic pathway for this molecule in microorganisms (i.e. yeast or E.coli) Applications: Medicine THERAPEUTIC BACTERIA • Strange idea, we think of bacteria to be associated with disease, not therapy TUMOR-KILLING BACTERIA • Creating a safe synthetic bacteria to be injected into the bloodstream • Travel to tumor, insert itself into cancer cell, produce tumour-killing toxin Applications: Food Products ACTIVIA • People are infecting themselves with bacteria • Can improve digestion • People like this! Applications: Energy Production BIOFUELS • Important issue worldwide • Plants biofuels • Plant biomass simple sugars • Fermented sugar energy Applications: Risks • Natural genome pool contamination • Synthetic products released in the environment should have a specific life span • Creation of deadly pathogens: bio-terrorism • Negative environmental impact • Global monitoring and tracking of synthetic products are necessary Overview 1. ~1 million bp synthetic genome 2. Synthetic genome was transplanted into a cell of a different subspecies – booted up! 3. Vast implications/uses for applied microbiology 4. Synthetic biology can reshape our lives and transfer our society 5. Important concerns regarding religion (playing with god) should be discussed and addressed Questions & Ethics Discussion Thank you for staying awake Discussion Points • What if a synthetic RNA can be designed to catalyze its own reproduction within an artificial membrane? • No guarantee that a synthetic genome that works for one organism (E. coli) will work in another (B. subtilis) • Cost/expenses • Religious/ethical issues References • Gibson, D. G., Glass, J. I., Lartigue, C., Noskov, V. N., Chuang, R., Algire, M. A., et al. (2010). Creation of a bacterial cell controlled by a chemically synthesized genome. Science, 329(5987), 52-56. • Lartigue, C., Glass, J. I., Alperovich, N., Pieper, R., Parmar, P. P., Hutchison III, C. A., et al. (2007). Genome transplantation in bacteria: Changing one species to another. Science, 317(5838), 632-638. • Laitigue, C., Vashee, S., Algire, M. A., Chuang, R. -., Benders, G. A., Ma, L., et al. (2009). Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science, 325(5948), 1693-1696.