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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 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 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 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 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 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. Essential Genes – A Comparative Study 450 400 Essential Genes 350 300 Craig Venter, 2005 250 Ehrlich SD,2003 200 Gil,2004 Koonin,1996 150 100 50 0 Studies on Minimal Genome 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 Functional Groups Main Roles Intermediary metabolism Transport and binding proteins Protein fate Transcription Cell envelope Hypothetical proteins Unknown function Nucleosides and nucleotides Energy metabolism Protein synthesis cell/organism defense Synthesis of cofactors and carriers Cellular processes Fats and phospholipid metabolism Regulatory function DNA metabolism Craig Venter, 2005 4 35 Ehrlich SD, 2003 2 35 Koonin, 1996 0 7 Gil, 2004 24 12 35 47 37 16 29 95 1 7 16 7 35 47 36 8 21 11 1 2 3 1 23 43 20 1 1 7 1 1 10 3 35 45 29 8 10 10 1 3 6 6 5 3 0 2 2 5 2 25 2 12 1 3 2 11 2 32 Comparison of Functional Groups 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. Designing a Synthetic Organism STRATEGY Antisense RNA Computationally Predicted Engineer the genome/add new functions Transposon Mutagenesis Determine the minimal genes Gene knockout using non replicating plasmid insertions Synthesize and assemble the genome Genome Transplantation SYNTHETIC ORGANISM Into a suitable propagating cell that can take up the genome Success so far… Infectious Virus Completely Synthesized – World’s First Artificial Organism - 2002 3026 bp 1895 bp 2682 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 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 Proposed Applications 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 . 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 ? Biofuel – A Strategy for Designing synthetic organism Synthetic organism which produces ethanol with minimal genes Identified Essential gene list Adh,pdc, pfl Add new genes to the existing prototype and assemble genome Genome Transplantation NO YES What next ? Add few more imp genes success Screen for viability of cell , maximum replication and higher ethanol production 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. 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) Inactive form Disease Indicator s Active form Programmed Module Therapeutics Genetic code expansion 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 The Hindering Factor 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 How to overcome ? 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 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. 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 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 Thank You!