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Synthetic Biology and Whole-Cell Technologies

This article explores the applications of synthetic biology and whole-cell technologies, focusing on genetic engineering, regulation, multi-gene circuits, and new synthesis methodology. It also discusses the concept of abstraction hierarchy in biological systems. The article includes examples such as Euprymna scolopes and symbiotic Vibrio fischeri, Agrobacterium infection, fruiting body formation by Myxococcus xanthus, microbial cellulose production by Acetobacter xylinum, and erythromycin biosynthesis by Saccharopolyspora erythraea.

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Synthetic Biology and Whole-Cell Technologies

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  1. Synthetic Biology and Whole-Cell Technologies J. Christopher Anderson

  2. Celebrities of the Prokaryotic World Applications Euprymna scolopes and symbiotic Vibrio fischeri Agrobacterium infection Fruiting Body formation by Myxococcus xanthus Microbial cellulose production by Acetobacter xylinum Erythromycin biosynthesis by Saccharopolyspora erythraea Oil bioremediation by Acinetobacter sp.

  3. Applications What is Synthetic Biology? Ground-up Genetic or Cellular Engineering, we add DNA sequences into well-characterized model organisms to understand biological processes or create useful organisms • Putting the Engineering back in Genetic Engineering • Focus on regulation and multi-gene circuits or networks • New, whole-cell technologies • New synthesis methodology and exploiting 1Mb of memory

  4. Abstraction and Standardization

  5. A Mechanical System: The Lunar Rover Systems On Earth In a rocket On the moon Automobile Chassis Human Transport Safety Sampling Communication Collapsability

  6. Synthesis The Abstraction Hierarchy Systems Processes Devices 1 Devices 2 Devices ...n Parts

  7. A Mechanical System: The Lunar Rover Systems

  8. The Enterobacterial Chassis Systems Cell growth and division The Central Dogma Primary Metabolism Environment Sensing • ~3000 genes common to all bacteria • Encoded by ~3 million bases of DNA • Almost identical among Enterobacteria

  9. Deletions in other enterobacteria Deletions Made in E. coli K12 MG1655 Mycoplasma genitalium Mesoplasma florum Blattner and coworkers. Science. 2006 May 19;312(5776):1044-6 Construction of Minimal Genomes Systems

  10. Type 1 Pili Lab E. coli K12 What Distinguishes Bacteria? Systems

  11. Type 1 Pili 2 TTSS Systems 4 Adhesion Systems Iron Acquisition Lab E. coli K12 Flagella 2 O-antigens Pathogenic Salmonella typhimurium (4,857,432 bp) What Distinguishes Bacteria? Systems

  12. Synthesis The Abstraction Hierarchy Salmonella typhimurium The System Processes Devices 1 Devices 2 Devices ...n Parts

  13. Synthesis The Registry of Parts Basic Parts Promoters Ribosome Binding Sites Open Reading Frames Terminators Devices A GFP Producing Device tetR RBS GFP Ter. Ter.

  14. Synthesis The Biobrick Standard

  15. Gene Synthesis Synthesis

  16. Synthesis The Abstraction Hierarchy Systems Processes Devices 1 Devices 2 Devices ...n Parts

  17. Information Carriers

  18. Electronic Signal Carrier Devices Systems Sensors Processors Actuators

  19. Enzymes IppHp IspA AtoB HMGS tHMGR Mevalonate Acetyl-CoA AA-CoA HMG-CoA MK PMK MPD Idi IspA DMAP IPP Mev-P Mev-PP ADS CYP7AV1/CPR Artemisinic Acid Artemisinin FPP Amorphadiene Chemical Information: Biosynthesis of Artemisinin Systems E. coli Yeast Artemisia annua Keasling and coworkers. Nature. 2006 440(7086):940-3., Nat Biotechnol. 2003 21(7):796-802.

  20. Chemical Information: Biosynthesis of Artemisinin Systems Isoprenoid Biosynthesis FPP Amorphadiene Biosynthesis Amorphadiene Artemisin Biosynthesis Artemisinin

  21. Chemical Information: Biosynthesis of Artemisinin Systems Isoprenoid Biosynthesis Exogenous FPP FPP Amorphadiene Biosynthesis Amorphadiene Artemisin Biosynthesis Artemisinin

  22. Chemical Information: Biosynthesis of Artemisinin Systems Isoprenoid Biosynthesis FPP FPP Amorphadiene Biosynthesis Taxol Biosynthesis Amorphadiene Taxol Artemisin Biosynthesis Artemisinin

  23. The Transcription Paradigm Systems Signal Processors Sensors Actuators

  24. The Transcription Paradigm Systems Signal Processors Sensors Actuators ENVIRONMENT Sensors TRANSCRIPTION

  25. The Transcription Paradigm Systems Signal Processors Sensors Actuators TRANSCRIPTION Processors TRANSCRIPTION

  26. The Transcription Paradigm Systems Signal Processors Sensors Actuators TRANSCRIPTION Actuators Changes in phenotype or biochemistry

  27. Sensors

  28. The Light Sensor Levskaya et. al. Nature. 2005 Nov 24;438(7067):441-2.

  29. The TNT Sensor Hellinga and coworkers. Nature. 2003 May 8;423(6936):185-90

  30. P OmpR OmpR Sensing by Histidine Kinases OM IM EnvZ OmpC Promoter Conversion of Black Pigment LacZ

  31. OmpR Sensing by Histidine Kinases OM TNT Light Aspartate BP Trg CphI Tar IM EnvZ EnvZ EnvZ EnvZ Taz • Promoter ompC is an intrinsic sensor • LacZ is an Actuator ompC Promoter LacZ

  32. Intrinsic and Extrinsic Sensors Intrinsic Sensors in E. coli Stimuli Promoter Osmolarity ompC Copper cusS Arabinose araB Lactose lacZ Low oxygen fdhF High oxygen cyoA low pH yghD low Magnesium mgrB Low Temp. hybB Envelope stress degP Stationary phase katE Extrinsic Sensors Stimuli Promoter Light Engineered TNT Engineered High density Vibrio fischeri Salicylate Pseudomonas putida

  33. Processors

  34. Logic Gates NOT (Inverters) IF (Amplifiers) OR NOR AND TetR Promoter OUT Promoter IN tetR

  35. Analog to Digital Converter All-or-None Signal Out Graded Signal In FimE Promoter OUT Promoter IN Actuator F F fimE Ham et al. Biotechnol Bioeng.2006 May 5;94(1):1-4.

  36. Band Filter LacI LacIM1 Promoter IN Promoter OUT lacI cI LacI cI Weiss and coworkers. Proc Natl Acad Sci U S A.2004 101(17):6355-60.

  37. Actuators

  38. Reporter Genes GFP Green Fluorescence RFP Red Fluorescence YFP Yellow Fluorescence lacZb-gal assays phoA Phosphatase assays CmR CAT Assays

  39. Biosynthesis Artemisinin Erythromycin Ethanol Polyhydroxybutyrate Polyphosphate Cellulose Enzymes Antibodies Insulin Human growth hormone

  40. Behavior Actuators tra Conjugation (transfer of plasmids between cells) fim Type I Pili (adhesion to animal cells) inv Invasin (invasion of mammalian cells) fla Flagella (chemotaxis apparatus for swimming) csg Biofilm Formation

  41. Processes

  42. Artemisinin Revisited Lac Promoter tHMGR HMGS atoB Sensing Processing Actuation Lac Sensor Artemisinin Producer Nothing

  43. Radiation-Dependent Biofilms Sensing Processing Actuation Radiation Sensor Toggle Switch Biofilm Producer Collins and coworkers. Proc Natl Acad Sci U S A.2004 Jun 1;101(22):8414-9.

  44. Pattern Formation-a simple staged system Sensing Processing Actuation aTc Sensor AHL Producer Nothing AHL Sensor Band Filter GFP Producer Weiss and coworkers. Nature. 2005 Apr 28;434(7037):1130-4.

  45. Synthesis The Abstraction Hierarchy Systems Processes Devices 1 Devices 2 Devices ...n Parts

  46. Specialty Chemicals Therapeutics Bioremediation Agents Biopolymers Materials • Compactnanoscalesensor, controller, actuator packages • Highly engineerable • They are self-assembling and self-replicating • Derived from cheap, renewable resources Biosensors

  47. Whole-Cell Technologies are Cheap to Produce Applications 10¢/gram

  48. Applications Very Smart Drugs Really smart drugs

  49. History of Bacterial Therapeutics Applications Digestive Disorders Bladder Cancer Hair Removal/Restoration Nail Fungus GI Parasites Autoimmune Diseases Viral Infections Arteriosclerosis Solid Tumors

  50. Treating Cancer with Bacteria Applications • Cancer Immunotherapy • Localization to and killing of cancer cells

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