Synthetic Biology Research at the Office of Naval Research (ONR)

1. Synthetic Biology Research at the Office of Naval Research (ONR) Linda A. Chrisey, Ph.D. Program Officer, Naval Biosciences & Biocentric Technology Linda.chrisey@navy.mil

2. For Official Use Only

3. “Team Bio” • Warfighter Performance Department • Dr. Linda Chrisey (341) • Dr. Laura Kienker (342) • Dr. Tom McKenna (341) • Plus Collaborations with ONR Codes: • 31 (Command, Control Communications, Computers, Intelligence, Surveillance, and Reconnaissance), • 33 (Sea Warfare and Weapons), & • 35 (Naval Air Warfare and Weapons)

4. Vision: Identify and exploit key principles from Nature to design, control and power autonomous systems; to provide improved processes, materials and sensors; and to develop synthetic biology tools and applications to support the future Naval force. S&T Goals: 1. Design, control, and powering of Bio-inspired Autonomous and Surveillance Systems for improved stealth, maneuver-ability and mission capability 2. Elucidate and exploit Bio-inspired Processes, Materials and Sensors to provide enhanced system-level performance, lower cost, and greater security/versatility in operations 3. Explore the promise of Synthetic Biology to support the future Naval force with agile and organic approaches for surveillance, autonomous systems, and materials production

5. S&T Goal #3: Explore the promise of Synthetic Biology to support the future Naval force with agile and organic approaches for surveillance, autonomous systems, and materials production

6. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force • Rationale for this Goal: Current approaches for naval material manufacturing, environmental or biomedical sensing, and micro-device control are sub-optimal due to feedstock limitations or insecurity, sensors that detect but do not respond to threats, and “dumb” robotic devices/systems. • Synthetic Biology offers new, modular tools and approaches to design or modify organisms for specific functions. • Organisms may be programmed in this way, to deliver: • living material ‘factories’, • stealthy and distributed ‘sentinels’ that detect AND respond to threats, and • “brain-like” control of nano-/ micro-devices. • This goal aims to develop transformational approaches to multiple naval application areas using living organisms, to produce, deliver, detect and/or respond to compounds, or to provide C2 to non-living devices • Impact if we attain this Goal: • Secure, renewable, scalable production of high-value naval materials. • Biomedical compounds delivered to the right place and the right time. • Stealthy, remotely-observable sentinel species for threat monitoring • Autonomous, living-non-living integrated micro-devices

7. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force • Sub-Goal 3.1 : Develop Secure And Sustainable Bio-based Production of naval materials • Approach: Metabolic engineering of bacteria or plants to use renewable feedstocks (light+ CO2, sugars, stover) to produce and/or secrete energetic or other materials (i.e., fuels). • PO Involved: Kienker, Chrisey

8. S&T Goal: Naval Materials and Electronics - Secure and Sustained Production …from biomolecules to biomimetic systems Butanetriol Trinitrate (BTTN) (Plasticizer used in Propellants & Explosives) Chemical Butanetriol (BT) Nitration Bio-nitration In future Sugars Microbial Triamino-trintrobenzene (TATB) (Used in Fuse & Booster Systems) Phloroglucinol (PG) Chemical Nitration Microbially Synthesized Energetic Materials Sub-goal: Secure And Sustainable Bio-based Production S&T Aims: Metabolically engineer bacteria and plants to manufacture energetic materials (EM) for explosives and propellant formulations (i.e. butanetriol trinitrate (BTTN) and triaminotrinitro-nbenzene (TATB)). 1) Implement green syntheses of EM cores for conventional chemical nitration (i.e. butanetriol (BT) and phloroglucinol (PG), respectively). 2) Implement green bionitration systems to eliminate chemical nitration. Rationale: Conventional petroleum-based chemical syntheses of BT and PG are environmentally harmful, costly, and rely on insecure feedstock materials. Currently, no U.S. manufacturer of BT or PG. Impact: Domestic production of BT and PG becomes possible. → ensures availability of BTTN & TATB to the Navy → may expand uses of BT and PG which would reduce production costs of TATB and BTTN PO Involved: Kienker (laura.kienker@navy.mil)

9. S&T Goal: Naval Materials and Electronics - Secure and Sustained Production …from biomolecules to biomimetic systems • Sub-goal: Secure And Sustainable Bio-based Production(con’t) • Accomplishments: • Feasibility of bacterial synthesis of the BT and PG from renewable agricultural feedstocks demonstrated. • Discovered a new class of enzyme, a nitrososynthase, responsible for alkylnitrosugar formation in natural product • Bacterial genes for PG production cloned into plants; verification of synthesis in vivo in progress • Demonstrated that covalent and non-covalent enzyme assemblies promote efficient metabolite biosynthesis.

10. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force • Sub-goal: Secure And Sustainable Bio-based Production • S&T Transitions: • FY06: Biosynthetic D-BT tested by NSWC-IH and found useful for D-BTTN production • FY06: Draths Corp. founded by ONR-funded PI for microbial synthesis of D-BT and PG. • FY07: SERDP (6.3) funds Draths Corp. and ATK-Thiokol to manufacture TATB and TNT from biosynthesized PG. • FY08: ESTCP funds NSWC-IH and Draths Corp. to dem/val the microbial syntheses of D-BT and PG. (Draths Corp. backed out on participation 6 months into award.) • FY09: Commercialization of microbially synthesized PG planned by Draths Corp. • Phytodetectors, Inc. to develop/commercialize ‘sense & respond’ plants (2007) • TSWG, DHS, CIA all briefed on plant TNT-detector technology (2008) • AFRL co-funding remote detection studies via Phase II STTR (ongoing) • Potential Customers: NSWC-IH, U.S. Army, ATK-Thiokol, TSWG, JIEDDO, NAVSEA, SOCOM, NAVFAC, Navy Fuels IPT, Navy Depots, Industry

11. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force Sub-Goal 3.2:Develop genetically EngineeredSentinel Organisms for threats, i.e., explosives or pathogens. Approach: Characterize natural bacterial/cellular responses to environmental signals/stimuli at a genetic level. In silico design, construct, and evolve functional organisms that detect and respond to specific stimuli of interest. PO Involved: Chrisey

12. S&T Goal: Design, Control and Powering of Surveillance Systems …from biomolecules to biomimetic systems • Sub-goal: Engineered Sentinel Organisms • S&T Aims: • Develop genetically engineered organisms that can sense and respond to threats, i.e., explosives. • Explore use molecular and synthetic biology tools for creating flexible receptor design and signal response components (e.g., non-natural functions). • Rationale: Smart sentinel” organisms offer increased stealth as well as capability to respond to or mitigate a threat (e.g., counter IED). “ Plug-and-play” genetic/ metabolic engineering of cell or plant platforms permits agile responses to changing threats • Impact: Distributed and stealthy persistent environmental surveillance possible at the macro-scale (plant) or micro-scale (diatoms, algae, bacteria). Potential for remote surveillance (hyperspectral imaging), even in challenging environments (e.g., littoral-to-beach zone) • PO Involved: Chrisey (linda.chrisey@navy.mil) Visible Remote 0 24 48 72 0 24 48 72 Control Leaf Assays Preliminary Screens (TNT Ligand)

13. S&T Goal: Design, Control and Powering of Surveillance Systems …from biomolecules to biomimetic systems Staphylococcus, Bacillus & Streptococcus AIP Programmed Sentinel/Killer cells • Sub-goal: Engineered Sentinel Organisms (con’t) • Accomplishments: • Terrestrial plant engineered to specifically detect and respond to TNT. Plant “de-greens” at [TNT] as low as 23 ppTr (2006) • Bacteria engineered to detect edges of light fields (2009) • Diatom engineered with bacterial ribose binding protein (silaffin fusion, 2010) • E. coli stimuli-classifying promoters characterized (2009) • Engineered modular sentinel circuits in E. coli and mammalian cells for the detection and destruction of several gram negative and positive pathogenic bacteria • Characterization of magnetic field detection/magnetotaxis in bacteria • Potential Customer: TSWG, JIEDDO, NAVSEA, SOCOM, NAVFAC • Transitions: • Colorado State U received $8M DTRA funds for plant tech for CW agent detection • Phytodetectors, Inc. to develop and commercialize ‘sense and respond’ plants (2007) • AFRL co-funded remote detection studies via Phase II STTR

14. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force • Sub-Goal 3.3: Support Synthetic Biology Tool Development to enable rapid multi-gene pathway design and synthesis; novel receptors: validated genetic circuit control elements; and desired functional pathways • Approach: Utilize novel, rapid methods to simultaneously evolve or design multiple pathways to attain desired function, improve long multi-gene DNA synthesis strategies, and fully characterize and test known and evolved genetic regulatory elements.

15. S&T Goal: Design, Control and Powering of Surveillance Systems …from biomolecules to biomimetic systems • Sub-goal: Syn Bio Tool Development • S&T Aims: • Develop cost-effective, high-throughput methods for rapid genome or phenotype engineering. • Design and validate a set of simple genetic logic gates that can be assembled into more complex genetic circuits that integrate information from multiple sensors • Extend biophysical models that map the sequence of a genetic part to its function. • Rationale: Time and cost of preparing and testing multi-gene sequences is high. Current genetic circuit programming limited to small number of well-validated parts which limit design of novel, yet predictable, functional systems • Impact: Improved methods for design of function-specific organisms. • PO Involved: Chrisey (linda.chrisey@navy.mil)

16. S&T Goal: Design, Control and Powering of Surveillance Systems …from biomolecules to biomimetic systems • Sub-goal: Syn Bio Tool Development (con’t) • Accomplishments: • Published a mathematical model that calculates the strength of a ribosome binding site (RBS calculator) • Developed a method for highly parallel chip-based gene synthesis [50-fold enhancement in assembled multi-gene sequence length (35 kilobase pairs)]. • “Rapidadaptor” method for preferential mutation of F’-episomal genes over chromosmal genes (allows for mutation of specific genes in a test organism and concurrent testing of those mutations • Potential Customer: Industry, potentially NAVFAC, SOCOM, MANTECH • Transitions: • RBS calculator licensed to Dow Agriculture, Bayer Crop Science, DSM, Life Technologies, and Genomatica • Formal collaboration between Wyss Inst and Agilent Technologies to create an automated platform for high-throughput gene synthesis utilizing unique multi-gene assembly approach

17. S&T Goal 3: Develop Synthetic Biology Tools and Applications to Support the Future Naval Force • Sub-Goal 3.4: Develop Bacterial or Cell-based Controllers or “brains” for C2 of micro-devices • Approach: Explore methods (other than electrical) for transduction of environmental signals and responses by bacteria/cells through interfaces to devices. Translate cellular behaviors such as chemotaxis, phototaxis, magnetic detection, etc to mimicked action by non-living micro-device. • Newly funded effort – 2011 Multi-disciplinary University Research Initiative (MURI) – “Utilizing Synthetic Biology to Create Programmable Micro-Bio-Robots “ (Collins, Boston U)

18. S&T Goal: Design, Control and Powering of Surveillance Systems …from biomolecules to biomimetic systems • Sub-goal: Bacterial or Cell-based Controllers • S&T Aims: • Create programmable bacteria capable of detecting and processing a wide range of environmental signals • Develop light-based and chemical-based systems that enable two-way interfaces and communication between bacteria and • robot systems • Create micro-bio-robots (MBR’s) s by integrating programmable bacteria with non-living platforms and novel biomaterials. • Develop sophisticated, macro-scale underwater “chaperone” robots to interact with MBRs, and algorithms to control heterogeneous team of MBRs and chaperone robots • Rationale: Living cells process and respond to multiple types of stimuli (chemical, EM, mechanical…). Can we increase capabilities of robots by giving them living cell “brains”? • Impact: Reduce sensor payloads/power demands on robots while increasing capabilities • PO Involved: Chrisey (linda.chrisey@navy.mil) Underwater macrorobots to be coordinated with micro-bio-robots

19. Summary