Synthetic Biology-Engaging Biology with Engineering
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Synthetic biology is an interdisciplinary field that applies engineering principles to design and construct new biological systems. It has enabled the design of novel organisms through DNA synthesis and assembly. Key technologies like DNA sequencing and synthesis have accelerated progress in synthetic biology. The field utilizes standard biological parts and computational tools to rationally design and model new systems. Promising applications include production of fuels, drugs and materials. However, synthetic biology also raises social and ethical challenges regarding biosafety, biosecurity and naturalness that warrant careful consideration and oversight.
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Synthetic Biology-Engaging Biology with Engineering
- 1. SYNTHETIC BIOLOGY : ENGAGING BIOLOGY WITH ENGINEERING Student Navaneetha Krishnan J II M.Sc (Biotechnology) Chairman Dr. M. Bharathi Professor, DPMB&B, CPMB&B.
- 2. OUTLINE • INTRODUCTION • BRIEF HISTORY OF SYNTHETIC BIOLOGY • APPROACHES FOR SYNTHETIC BIOLOGY • iGEM AND SYNTHETIC BIOLOGY • DNA ASSEMBLY FOR SYNTHETIC BIOLOGY • CHASSIS FOR SYNTHETIC BIOLOGY • COMPUTATIONAL TOOLS GUIDING SYNTHETIC BIOLOGY • POTENTIAL APPLICATIONS OF SYNTHETIC BIOLOGY • SOCIAL AND ETHICAL CHALLENGES • CASE STUDIES
- 3. INTRODUCTION • Synthetic biology is the design and construction of new biological parts, devices, and systems and the re-design of existing biological systems (Arkin et al., 2009). The need for synthetic biology biologists chemists engineers
- 4. Synthetic biology – an interdisciplinary science http://www.synthetic-biology.info/synbio.html
- 5. Is synthetic biology achievable? YES. Due to the following aspects of biology : (1) biology is hierarchical and, (2) biology re-uses a small set of simple parts to create complex behaviors. • Synthetic biologists, design new biological systems having in mind the top of hierarchy, but actually operating at the bottom of the hierarchy, by designing and testing novel gene and protein combinations, for how the smallest parts (genes and the proteins they encode) are wired. Peisajovich et al., 2007
- 6. Technologies enabling rapid developments in synthetic biology • DNA synthesis and assembly • Gene sequencing • ‘Omics’ technologies • Bionformatics and Computational biology Biolytic's Dr. Oligo - DNA synthesizer Illumina Hiseq 2000
- 7. SYNTHETIC BIOLOGY : A BRIEF HISTORY Cameron et al., 2014
- 8. contd… Cameron et al., 2014
- 9. Synthetic biology - hierarchy Andrianantoandro et al., 2006
- 10. Employing engineering principles in synthetic biology Endy et al., 2007; Andrianantoandro et al., 2006 Standardization Abstraction Decoupling
- 11. Synthetic biology design process Marguet et al., 2007
- 12. Synthetic biology – Systems biology linkage Federici et al., 2013
- 13. APPROACHES IN SYNTHETIC BIOLOGY Silver and Way, 2014
- 14. iGEM - International Genetically engineered Machines Competition TOM KNIGHT http://www.technologyreview.com/article/423703/rewiring-cells/ Christina D Smolke, 2009
- 15. Registry of Standard Biological parts http://parts.igem.org/Catalog
- 17. iGEM 2008 – IIT Madras Team
- 18. iGEM 2014 – IIT Delhi Team
- 19. DNAASSEMBLY FOR SYNTHETIC BIOLOGY Ellis et al., 2011
- 20. DNA assembly methods for synthetic biology Ellis et al., 2011
- 22. Circular polymerase extension cloning(CPEC) Quan and Tian, 2009
- 23. SLIC method Itaya et al., 2008
- 24. CHASSIS FOR SYNTHETIC BIOLOGY • Escherichia coli • Bacillus subtilis • Saccharomyces cerevisiae • Cell free protein synthesis system • Chlamydomonas reinhardtii • Geobacillus sp. • Marchantia polymorpha • Pichia pastoris • Synechocystis sp. • Synthetic Yeast 2.0 • Physcomitrella patens Established chassis Emerging chassis Kelwick et al., 2014
- 25. COMPUTATIONAL TOOLS GUIDING SYNTHETIC BIOLOGY Kelwick et al., 2014
- 26. contd… Kelwick et al., 2014
- 29. Potential applications Redesign through modification of existing pathways • Succinate • Malate • D-lactate • Acetate • Butanol Redesign through introduction of foreign or non-natural pathways • Ethanol • L-lactate • Xylitol • L-Alanine • Lycopene • Paclitaxel Jarboe et al., 2010; Li and Pfeiffer, 2014 Metabolic engineering
- 30. Synthetic Genomics Inc http://www.syntheticgenomics.com/index.html Industry focus J. Craig venter
- 31. Biomedicine • Synthetic biology has the potential to engineer novel diagnostic and therapeutic strategies for relatively intractable medical conditions like cancer and infectious diseases. Drug Discovery and Production • Artemisinin : Antimalarial drug Vaccine Development • Synthetic vaccines and antibodies. Treatment of infectious diseases • Treatment of bacterial infections by commensal bacteria. • Treatment of bacterial infections by engineered bacteriophages. • Sequence-Specific Endonucleases for Disruption of Bacterial and Viral Infection. Zhao et al., 2014
- 32. Inovio SynCon® Vaccines: from Bug to Vaccination http://www.inovio.com/technology/synthetic-vaccines/ Industry focus
- 33. tSVP™ - A new class of synthetic vaccines for Optimal Immune Response http://selectabio.com/product-platform/ Industry focus
- 34. Engineering genomes Gibson and Venter, 2014
- 35. Biomaterials • Discovery of novel biomaterials and cell-based synthesis of useful materials. • Use of DNA nanotechnology for accurate construction of in vitro nanopatterns that can serve as scaffolds for biomaterials. • New scaffolds for tissue engineering,enhanced surgical materials and biocompatible device coatings for medical applications. Cheng and Lu, 2012
- 36. • First report of production of a synthetic cell – “Syn 1.0” • Genome of a bacterial species synthesised artificially and assembled in the Yeast and subsequently transplanted in to another related bacterial species. • Donor bacterium : Mycoplasma mycoides Recipient bacterium : Mycoplasma capricolum • The synthetic bacterium had expected phenotypic properties and capable of continuous self replication.
- 37. • Synthetic genome was designed based on finished genome sequences of two laboratory strains of M.mycoides subspecies capri GM12 .One developed by Lartigue et al (CP001621) and other YCpMmyc1.1(CP001668). Synthetic genome assembly strategy 1078 casettes (each 1,080 bp (~1kb) in length (having 80 bp overlaps to adjacent cassetes) 10kb synthetic intermediates (each containing 10 no:s of 1kb cassetes) (111 no:s) 100 kb synthetic intermediates(11 no:s) (recombined in yeast) Circular genome Genome transplantation in M.capricolum
- 39. Fig. 1 The assembly of a synthetic M. mycoides genome in yeast. D G Gibson et al. Science 2010;329:52-56 Published by AAAS
- 40. Fig. 2 Analysis of the assembly intermediates. D G Gibson et al. Science 2010;329:52-56 Published by AAAS
- 41. Fig. 3 Characterization of the synthetic genome isolated from yeast. D G Gibson et al. Science 2010;329:52-56 Published by AAAS
- 42. Fig. 5 Images of M. mycoides JCVI-syn1.0 and WT M. mycoides. D G Gibson et al. Science 2010;329:52-56 Published by AAAS
- 43. Two-dimensional gels were run using cell lysates from M. mycoides YCpMmyc1.1 (WT) and M. mycoides JCVI-syn1.0
- 44. Results and discussion • A single transplant from sMmYCp235 synthetic genome was sequenced. • This strain was referred to as M.mycoides JCVI-syn1.0 • Sequence of syn1.0 compared with YCpMmyc1.1 • 8 new SNPs,an E.coli transposon insertion and 85bp duplication. • No sequences related to M.capricolum found in the transplant.
- 46. Overview of the yeast-based semi-synthetic process for the production of artemisinin
- 47. CJ Paddon et al. Nature 000, 1-5 (2013) doi:10.1038/nature12051 Artemisinic acid production pathway in S. cerevisiae and summary of strains described.
- 48. Production of crystalline artemisinic acid produced in shake flask cultures
- 49. Maximum artemisinic acid titer in fed-batch fermentation process
- 50. CJ Paddon et al. Nature 000, 1-5 (2013) doi:10.1038/nature12051 Increasing production of artemisinic acid by strain engineering and addition of IPM to cultures.
- 51. CJ Paddon et al. Nature 000, 1-5 (2013) doi:10.1038/nature12051 Growth, viability and production by S. cerevisiae strains.
- 52. CJ Paddon et al. Nature 000, 1-5 (2013) doi:10.1038/nature12051 Chemical conversion of artemisinic acid to artemisinin.
- 53. Social and Ethical challenges Synthetic Biology Economic risks : Intellectual property Environmental risks: Biosafety Social risks : Biosecurity Ethical issues : Natural/unnatural Source: International Risk Governance Council , Geneva, 2008
Editor's Notes
- The assembly of a synthetic M. mycoides genome in yeast. A synthetic M. mycoides genome was assembled from 1078 overlapping DNA cassettes in three steps. In the first step, 1080-bp cassettes (orange arrows), produced from overlapping synthetic oligonucleotides, were recombined in sets of 10 to produce 109 ~10-kb assemblies (blue arrows). These were then recombined in sets of 10 to produce 11 ~100-kb assemblies (green arrows). In the final stage of assembly, these 11 fragments were recombined into the complete genome (red circle). With the exception of two constructs that were enzymatically pieced together in vitro (27) (white arrows), assemblies were carried out by in vivo homologous recombination in yeast. Major variations from the natural genome are shown as yellow circles. These include four watermarked regions (WM1 to WM4), a 4-kb region that was intentionally deleted (94D), and elements for growth in yeast and genome transplantation. In addition, there are 20 locations with nucleotide polymorphisms (asterisks). Coordinates of the genome are relative to the first nucleotide of the natural M. mycoides sequence. The designed sequence is 1,077,947 bp. The locations of the Asc I and BssH II restriction sites are shown. Cassettes 1 and 800-810 were unnecessary and removed from the assembly strategy (11). Cassette 2 overlaps cassette 1104, and cassette 799 overlaps cassette 811.
- Analysis of the assembly intermediates. (A) Not I and Sbf I double restriction digestion analysis of assembly 341-350 purified from E. coli. These restriction enzymes release the vector fragments (5.5 and 3.4 kb) from the 10-kb insert. Insert DNA was separated from the vector DNA on a 0.8% E-gel (Invitrogen). M indicates the 1-kb DNA ladder (New England Biolabs; NEB). (B) Analysis of assembly 501-600 purified from yeast. The 105-kb circles (100-kb insert plus 5-kb vector) were separated from the linear yeast chromosomal DNA on a 1% agarose gel by applying 4.5 V/cm for 3 hours. S indicates the BAC-Tracker supercoiled DNA ladder (Epicentre). (C) Not I restriction digestion analysis of the 11 ~100-kb assemblies purified from yeast. These DNA fragments were analyzed by FIGE on a 1% agarose gel. The expected insert size for each assembly is indicated. λ indicates the lambda ladder (NEB). (D) Analysis of the 11 pooled assemblies shown in (C) following topological trapping of the circular DNA and Not I digestion. One-fortieth of the DNA used to transform yeast is represented.
- Characterization of the synthetic genome isolated from yeast. (A) Yeast clones containing a completely assembled synthetic genome were screened by multiplex PCR with a primer set that produces 11 amplicons; one at each of the 11 assembly junctions. Yeast clone sMmYCp235 (235) produced the 11 PCR products expected for a complete genome assembly. For comparison, the natural genome extracted from yeast (WT, wild type) was also analyzed. PCR products were separated on a 2% E-gel (Invitrogen). L indicates the 100-bp ladder (NEB). (B) The sizes of the expected Asc I and BssH II restriction fragments for natural (WT) and synthetic (Syn235) M. mycoides genomes. (C) Natural (WT) and synthetic (235) M. mycoides genomes were isolated from yeast in agarose plugs. In addition, DNA was purified from the host strain alone (H). Agarose plugs were digested with Asc I or BssH II, and fragments were separated by clamped homogeneous electrical field (CHEF) gel electrophoresis. Restriction fragments corresponding to the correct sizes are indicated by the fragment numbers shown in (B).
- Images of M. mycoides JCVI-syn1.0 and WT M. mycoides. To compare the phenotype of the JCVI-syn1.0 and non-YCp WT strains, we examined colony morphology by plating cells on SP4 agar plates containing X-gal. Three days after plating, the JCVI-syn1.0 colonies are blue because the cells contain the lacZ gene and express β-galactosidase, which converts the X-gal to a blue compound (A). The WT cells do not contain lacZ and remain white (B). Both cell types have the fried egg colony morphology characteristic of most mycoplasmas. EMs were made of the JCVI-syn1.0 isolate using two methods. (C) For scanning EM, samples were postfixed in osmium tetroxide, dehydrated and critical point dried with CO2, and visualized with a Hitachi SU6600 SEM at 2.0 keV. (D) Negatively stained transmission EMs of dividing cells with 1% uranyl acetate on pure carbon substrate visualized using JEOL 1200EX CTEM at 80 keV. To examine cell morphology, we compared uranyl acetate–stained EMs of M. mycoides JCVI-syn1.0 cells (E) with EMs of WT cells made in 2006 that were stained with ammonium molybdate (F). Both cell types show the same ovoid morphology and general appearance. EMs were provided by T. Deerinck and M. Ellisman of the National Center for Microscopy and Imaging Research at the University of California at San Diego.