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DESIGNING A SYNTHETIC ORGANISM

DESIGNING A SYNTHETIC ORGANISM. Asfa A S (HT080934L) Vasanth Natarajan (HT081073M). Department of Chemical & Biomolecular Engineering National University of Singapore. Vision of “SYNTHETIC BIOLOGY “. Recreate Life. Origin of Life. SYNTHETIC BIOLOGY. Minimal Genome. Designer Cells.

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DESIGNING A SYNTHETIC ORGANISM

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  1. DESIGNING A SYNTHETIC ORGANISM Asfa A S (HT080934L) Vasanth Natarajan (HT081073M) Department of Chemical & Biomolecular Engineering National University of Singapore

  2. Vision of “SYNTHETIC BIOLOGY “ Recreate Life Origin of Life SYNTHETIC BIOLOGY Minimal Genome Designer Cells

  3. Synthetic Biology Synthetic biology is an ambitious and relatively new field of biology that hopes to recreate life. The first and foremost challenge in creating 'life in lab' lies in identifying the minimum essential components that can take on the essential properties of a living organism

  4. What Defines LIFE LIVING CELL Autonomous Replication Darwinian Evolution Continued growth and division dependent on input of small molecules and energy Genetic and phenotypic variation for survival and reproduction

  5. Current Strategies Bottom Up Approach Top Down Approach • Strip down the genes of an existing cell to bare minimum enough to sustain life • Semi-synthetic • Design a protocell • Synthesizing cell from scratch

  6. Synthesizing Life – Bottom Up Approach • RNA - store information • RNA – RNA polymerase – replicate its own sequence • 2 RNA molecules – simplest cell • Assembly of single lipid molecules/micelles • Gradual growth • Environmental factors to control division Szostak et al. , Nature 2001

  7. Minimal Genome Concept • Aims to strip down a present day bacterium to its minimum essential components pertaining to replication, transcription and translation machinery. • Understand the basic components of the cell that makes it living. • Provides a template genome that can be used to recreate life • A less complex cell that can be reliably modeled and engineered to meet our requirements.

  8. Essential Genes – A Comparative Study • Mycoplasma genitalium – 482 protein coding genes – smallest genome • Craig Venter,2005 – 382 protein coding genes + 5 paralogous families – Transposon mutagenesis • Ehrilch SD, 2003 – 271 essential genes in Bacillus subtilis – Gene knock out by non replicating plasmid • Gil, 2004 – 206 essential genes – Comparison of Endosymbioints - Predicted • Koonin, 1996 – 256 essential genes – Comparison of M.genitalium and H.influenzae - Predicted

  9. Functional Groups

  10. Comparison of Functional Groups

  11. Essential Genes – A Conclusive List • Different studies come up with a different number of essential genes. • Computation - Underestimates minimal genes - accounts only those genes that have been conserved in evolution. • Transposon mutagenesis - Over estimates the genes – Classifies genes that slow down growth as essential and essential genes that tolerate mutation as non essential. • Antisense RNA - limited success rates • Most mutants produced are single mutants – synthetic lethality may not be accounted Construction of a single cell with systematic combination of all the mutations in a single strain is beyond the scope of present day technology.

  12. Designing a Synthetic Organism STRATEGY Transposon Mutagenesis Antisense RNA Gene knockout using non replicating plasmid insertions Computationally Predicted Determine the minimal genes Synthesize and assemble the genome Engineer the genome/add new functions Into a suitable propagating cell that can take up the genome Genome Transplantation SYNTHETIC ORGANISM

  13. Success so far… • Infectious Virus Completely Synthesized – World’s First Artificial Organism - 2002 3026 bp 2682 bp 1895 bp • cDNA - T7 RNA polymerase promoter constructed from 3 overlapping DNA fragments. • Each fragment - overlapping 400-600 bp. • Each segment – 69 nt of + and – sequences • cDNA transcribed – Infectious RNA • Infection demonstrated in mice. SYNTHETIC RNA => TRANSLATED => REPLICATED =>ENCAPSIDATED INTO NEW COAT PROTEINS Cello et al. Science, 2002

  14. In the Future… • Mycoplasma laboratorium • Synthetic Genome • Only essential 382 genes • Complete synthesis, cloning and sequential assembly • Synthetic Algae • Biofuel • Synthetic Genomics • Immortal synthetic organism • Military Purpose – Pentagon • Self killing switch

  15. Proposed Applications

  16. Biofuel – A dream in the making • Goals • Seeks alternatives to fossil fuels • Sustainability • Cost reduction • Challenges • Microorganisms can be designed to make useful materials from renewable materials (Sustainability)  - to seek alternatives to fossil fuels. • In this case, designing a set of chemical pathways which allows conversion of natural or waste materials for the production of Biofuels .

  17. Biofuel – A dream in the making (contd.) • ZM - Z.mobilis • SC - S.cerevisiae • EC - E.coli • adh,pdc,pfl • Genes that are important for ethanol production • How to design a synthetic organism by adding new functions to the existing genome ?

  18. Biofuel – A Strategy for Designing synthetic organism Synthetic organism which produces ethanol with minimal genes Identified Essential gene list Adh,pdc,pfl NO YES What next ? Add few more imp genes Add new genes to the existing prototype and assemble genome success Screen for viability of cell , maximum replication and higher ethanol production Genome Transplantation

  19. Biomedical Applications Devices- For example, for tissue regeneration or tissue repair complex molecular devices can be developed. - Another example could be development of macromolecular assemblies to sense the damage in blood vessels and repair them.

  20. Novel Drug Release Technology • Smart Drugs -----> Synthetic molecular ensemble • Encapsulates drug in an inactive form. • Sensing disease indicators • The programmed module will make a decision • Activates the drug . (Active only in cells affected by disease) Active form Inactive form Programmed Module Disease Indicators

  21. Therapeutics Genetic code expansion

  22. Environmental Applications • Bioremediation: • Treatment of environmental contaminants via biological systems. • Rational modification of bacteria and other microorganisms to eliminate toxic waste from soil. • For certain chemicals for which clean up is difficult, novel organisms with specific wiring can be used. • Biosensing : • Detect biotoxins • Helps in detecting toxin levels in environment

  23. The Hindering Factor How to overcome ? Obstacles • Bio engineered systems remains noisy • Not easier to predict accurately how a new system will behave • Engineered organisms capable of self replication and evolution • Expensive , Unreliable and adhoc biological systems

  24. FORSEEN RISKS • Some of the risks are indefinable at present – we cannot anticipate certain risks at this early stage • Accidental release of harmful organism - Extinction of existing species - Endemic - Damaging/Disrupting the habitat ( Upset natural balance) • Purposeful Design and release of harmful organism – Bioterrorism • Bio-hacker culture

  25. Control Measures • To educate and train a responsible generation of bioengineers and scientists • Working with approved research facilities • Controls and regulations can be imposed on part suppliers (eg . screening of oligonucleotides ) • Strict laws and policies to be imposed. • Incorporating novel genetic codes for high risk organisms to avoid tampering.

  26. Conclusion • Synthetic Biology – Greatest existing challenge • Synthetic and semi-synthetic approaches. • Discerning the minimal genome enhances better understanding of cells • Engineered organism can be used for various applications in fields of biomedicine and environment • Potential risks and hazards not clear

  27. Key References • Cello J et al. Chemical Synthesis of Poliovirus cDNA: Generation of Infectious Virus in the Absence of Natural Template. Science(2002), 297 • Smith et al. Complete Chemical Synthesis, Assembly and Cloning of a Mycoplasma genitalium Genome. Science(2008), 319 • Koonin et al. A Minimal Gene Set for Cellular Life Derived by Comparison of Complete Bacterial Genomes. PNAS(1996),93 • Venter C.J et al. Essential Genes of a Minimal Bacterium. PNAS(2006),103 • Szostak et al. Synthesizing Life. Nature(2001),409 • Ehrlich SD et al. Essential Bacillus subtilis genes.PNAS(2003),100 • Gil et al. Determination of the Core of a Minimal Bacterial Gene Set. Microbiology and Molecular Biology Reviews(2004),68

  28. Thank You!

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