A heavy price was paid for molecular biologys obsession with metaphysical reductionism' It stripped

1
The Shifting Paradigm for Biology

• A heavy price was paid for molecular biologys
  obsession with metaphysical reductionism. It
  stripped the organism from its environment .
  shredded it into parts to the extent that a sense
  of the whole--the whole cell, the whole
  multicellular organism, the biosphere--was
  effectively goneOur task is to resynthesize
  biology... The time has come for biology to
  enter the nonlinear world.
• Carl Woese, MMBR 2004

Biology of Complex Systems is a National Science
Priority Agencies should target investments
toward the development of a deeper understanding
of complex biological systems Scientific and
technological breakthroughs are expected in
diverse areas such as environmental
management OSTP/OMB interagency guidance memo
for FY 2005
2
A Systems Approach is needed to Bring the Genome
to Life
Systems Biology studying biological systems by
systematically perturbing them (biologically,
genetically or chemically) monitoring the gene,
protein, and informational pathway responses
integrating these data and ultimately
formulating mathematical models that describe the
structure of the system and its responses to
individual pertubations (Ideker et al., 2001
Annu, Rev. Genom. Hum. Genet. 2343) Mission
Science Requires an inherent systems view.
3
Integrated Approach to Shewanella Biology
MR-1 Genome Sequence, Bioinformatics
Information Synthesis Interpretation
Linked measurements
Imaging AFM, EM, CLSM Immuno-EM
Computational Biology
Concepts Hypotheses
Physiology Metabolism
Cellular networks, Modeling
Controlled Cultivation
Proteomics MS 2-D gel
Data Analysis Integration

• ?
• Perturbation
• mutation
• cultivation

Gene Expression Microarrays Reporters
4
Knowledge of Microbial Processes and Communities
Can Lead to New Solutions for Global Change

• Recommendations
• To enhance microbiological solutions to global
  change challenges, strengthen and expand ongoing
  research efforts, and direct new resources for
  basic research programs that
• Integrate an understanding of microbiological
  processes at all organizational levels, from
  individual organisms to ecosystems
• (2) Discover, characterize and harness the
  abilities of microbes that play important roles
  in transformations of trace gases and various
  toxic elements.
• Implement policies that promote effective
  long-term research on the microbiology of global
  change

5
GTL Program and Facilities Responding to AAM
Recommendations

• To make progress, science should not accept the
  limitations placed on discovery by traditional
  methods, conventional approaches, or existing
  infrastructure.
• Although progress in microbial genomics is being
  made at a fantastic rate, availability of
  appropriate tools still places limits on
  research.
• Powerful, but expensive, modern equipment should
  be housed in community facilities, open to
  researchers who might not otherwise have access
  to these technologies.
• ______
• Microbiology in the 21st Century Where Are We
  and Where Are We Going? American Academy of
  Microbiology, 2004

6
GTL Program and Facilities Responding to AAM
Recommendations

• Currently, ability to predict the responses of a
  single microorganism from the sequence of its
  genome can best be described as feeble, and our
  ability to make predictions for an assemblage of
  multiple organisms is even weaker.
• The ecosystem predictive capability will come
  from detailed work that links an understanding of
  the genome to an understanding of gene
  expression, protein function, and complex
  metabolic networks.
• A deep genomic understanding of these integrated
  microbial processes will provide a better
  understanding of our planet, our interaction with
  it, and our ability to predict and influence its
  future behavior.
• Create centers that facilitate research
  community access to post genomic analytical
  capabilities
• ________
• The Global Genome Question Microbes as the Key
  to Understanding Evolution and Ecology, American
  Academy of Microbiology, 2004
• http//www.asm.org/ASM/files/CCPAGECONTENT/docfile
  name/0000026555/GENOMEweb.pdf

7
Microbes and Biotechnology for DOE Missions R
D is Critical

• Why Microbes?
• Microbes and microbial communities are the
• Foundation of the biosphere and the planets
  ability to sustain all life.
• Masters at capturing, storing, and transforming
  energy.
• Most abundant and biochemically versatile life
  forms possessing diverse and sophisticated
  capabilities
• Recent Discoveries have revealed the dominant
  role of microbes in earth systems processes
• Prochlorococcus is the dominant photosynthetic
  organism on the planet.
• Sequencing of the Sargasso Sea has revealed
  immense diversity
• Microbes exist in the deep subsurface and survive
  on redox couples unrelated to photosynthesis
• Microbes are catalyst for acid mine drainage

Improvements in cellulase molecular machine
function are needed to more efficiently convert
cellulose to glucose for conversion to ethanol.
Applications of microbial capabilities will
stimulate new biotechnology industries to put
these new processes into the marketplace.
Published in J. Am. Chem. Soc. 2002, 124,
11242-3
Bioenzymes have been captured in a ceramic
nanomembrane with improved function and
stability.
8
GTL Systems Biology Molecules to Cells to
Communities
Communities
Single cells
Populations
Subcellular location and dynamics of machines
Gene expression in individual cells
Who is expressing what, when, where under what
conditions?

• Programmatic goals of understanding
• Proteins and Molecular Machines
• Regulatory processes
• Complex Microbial Communities
• and, developing the necessary computational and
  information architectures

9
Science enabled by the production and
characterization of proteins and molecular tags

• Investigating functions of unknown and
  hypothetical genes from microbial and
  meta-genomes
• The flood of genomes, unculturable systems
• Understanding natural systems genomic potential
  and behaviors
• Oceans, terrestrial, deep subsurface
• Understand microbial community organization and
  function
• Investigating natural variations in critical
  processes and designing and optimizing
  functionality
• Hydrogenases, cellulases, reductases
• Binding, products, biophysical parameters
• Enabling new single molecular investigation
  techniques
• Tags for Protein-protein and protein-DNA
  interactions, e.g. monitoring molecular
  structure, regulatory interactions,
  post-translational modifications
• Quantification standards for proteomics techniques

10
The Unknown Gene Function Problem

• Hypotheticals account for gt50 of potential
  protein-coding regions
• Escherichia coli
• 4,400 genes, ½ characterized to any extent
• current rate of 10 IDs/month, complete in 20
  years!
• Hundreds of genome sequences available, nos.
  increasing
• Microbial population sequencing Sargasso Sea
  sequencing (Venter et al.) alone added 1.2
  million previously unknown genes
• JGI 2004 Prokaryotes 235 Mb Microeuks 80Mb
  Communities 340 Mb

11
1/3 of Hypothetical Genes are expressed in
Shewanella oneidensis MR-1

• - gene expression (microarrays) either a signal
  was detected or not.
• - protein expression gt 500 MS/MS runs, gt 2
  million files!
• 0 FP, identifications with gt90 confidence
  level
• Hypothetical ORFs expressed as both RNAs and
  proteins
• Original Total Total no. of expressed genes
  ()
• genome no. of Transcriptome Proteome Both
  
• annotation genes
• All predicted ORFs
• 4468 3564 (80) 2252 (50) 2082 (47)
• Hypothetical ORFs
• 1623 1223 (75) 592 (36) 538 (33)

7 Additional Strains of Shewanella being
Sequenced by JGI
12
Identifying Protein Function

• Bioinformatics
• Biochemistry
• Biophysics
• Structure
• Genetics (e.g., mutagenesis)
• Partners/interactions
• Co-expression patterns

GTL Facilities will Provide Necessary
Capabilities Scale
Facility I - generate hypothetical proteins for
biochemical screen etc. as part of annotation
for each genome sequenced, even for uncultured
organisms!
13
Natural Systems of Interest to DOE Predict
behaviors and understand impacts of manipulations
Mar. 3-4 DOE/GTL Missions Science Workshop
14
Biotech could be a significant source of H2 in a
stabilization regime.
Total US End-Use Energy in 2005
Direct biological production of H2.
Jae Edmonds, JGCRI
15
BioH2 Production

• The development of methods for large-scale, i.e.
  hundred of exajoules of energy per year,
  cost-effective source of H2 using biotechnology
  could lower costs of addressing climate change by
  trillions of dollars!
• If H2-using technology (e.g. fuel cells) can be
  made cost effective
• BioTechnology could become a major source of
  energy
• Lower land-use emissions
• Lower agricultural prices.

Jae Edmonds, JGCRI
16
Hydrogenase Research Biophotolytic Hydrogen
Production
17
Bacteriorhodopsins via Ocean Metagenomics

• Rhodopsins are light absorbing pigments
  consisting of membrane proteins retinal
• Discovery of new rhodopsin via genome analysis of
  marine bacterioplankton (Béjà et al. 2000)
• Protein expressed in E. coli, bound retinol
  formed an active light-driven proton pump
• 782 new rhodopsin-like photoreceptors,
  representing 13 distinct subfamilies, identified
  in Sargasso Sea sequence (Venter et al. 2004)
• Do all variants absorb light? Structure-function
  relationships?

18
Cellulosic Ethanol Processing Plant in a
Microbe?
Today we utilize food starch to make alcohol and
complex and costly processing of
cellulose Tomorrow we want to utlitize high
yield cellulose crops with integrated processes
in microbes to convert to alcohols and other fuels
Cellulose Today
Decrystallization Hydrolysis of Cellulose,
Hemicellulose, and Lignin Multiple Sugar
Metabolism Alcohol Synthesis
Tomorrow?
19
Roadmap for Microbial Cellulosic Ethanol
Production Today Interim Goals
Endpoint
20
Molecular Tags for Single Molecule Methods

• A much more direct, sensitive and quantitative
  way to assess protein-DNA and protein-protein
  interactions (compared to yeast 2-hybrid,
  protein chips and mass spectrometry)

• GTL facilities provide an opportunity to bring
  this technology to high throughput performance
  (relatively large engineering effort)
• proteins, tags
• Instrumentation, data capture analysis

21
Single-Molecule Methods for the Characterization
of Protein-Protein Interactions and Expression
Levels in Shewanella oneidensis MR-1
Natalie R. Gassman1, Achillefs N. Kapanidis1,
Nam Ki Lee1,, Ted A. Laurence1,, Xiangxu Kong1,
and Shimon Weiss1,2,3 1Dept. of Chemistry and
Biochemistry, UCLA, 2Dept. of Physiology, David
Geffen School of Medicine, UCLA, 3California
NanoSystems Institute, Presenter
ngassman_at_chem.ucla.edu, presently Seoul National
University, presently Lawrence Livermore
National Laboratory
22
SORTING MOLECULES USING E, S
FRET-efficiency ratio (E)
(CONFORMATION INFO)
Fluorescence-aided molecule sorter
FAMS
Kapanidis et al., PNAS 101, 8936 (2004)
23
Protein-DNA InteractionDNA - Catabolite
Activator Protein (CAP) interaction
24
Protein-Protein Interactions of Transcription
Regulation
Two-component signal transduction, which
regulates transcription by the alternative sigma
factor, s54, provides a diverse array of
protein-protein and protein-DNA interactions to
assay using single molecule methods. Our focus
will be on the two-component system involving
nitrogen regulatory protein (NtrC).
25
Biological processes that can be dissected by
simultaneous monitoring of structure and
interactions
26
Purification of NtrC
Protein Production is a Bottleneck for HT Single
Molecule Methods!
Overexpression
Affinity Chromatography
27
GTL Program - Microbial Community Analyses
Cross-Cutting Processes
Microbial Communities
Marine
Soil
Subsurface
Photolytic H2 generation
Cross cutting processes/genes of Interest
Contaminant transformations
Biomass conversion
Example Analyses of Mission relevant Natural
Communities and Optimization of Processes require
a minimum production of 20,000 proteins per year
28
DOE Missions Tie to Capabilities for Microbial
Systems
Ocean
Natural Systems Behavior
Terrestrial
Subsurface
Toxic Metal Reduction
Convert Cellulose to Fuels
Energy
Convert Sunlight to Hydrogen
29
We can Achieve Dramatic Improvements in Quality,
Throughput, and Cost

• Genome projects demonstrated the power of
  high-throughput technologies, computational
  analyses, and data made available to all
  scientists.
• This approach can provide the needed gains for
  GTL biology.
• GTL pilot studies give us confidence in the
  technologies.
• Initial GTL systems biology research has
  identified key issues and requirements.
• The Approach
• Multidisciplinary teams,
• Scaleup and automation, focus on production
• Drive to dramatic production gains cost
  reductions
• Strict standards and methods for quality
  improvements,
• Characterization with advanced technologies such
  as synchrotron methodologies,
• Informatics and computing,
• Rapid and open availability of results to
  scientists -- freeing scientists from production
  activities

The learning curve for sequencing (above) is
being repeated for soluble and membrane proteins
(below)
30
GenomicsGTL Facilities - Enabling Cellular
Systems Science
Cellular Activities Understanding how cells
respond to environmental cues
Cellular Components Providing the basis for
determining protein function and cellular
processes
Developing a predictive understanding of the
functions of cells and communities of cells
Understanding how molecular machines are formed
and how they function
A New Infrastructure for Biological Research
31
Facility for Production and Characterization of
Proteins and Molecular Tags

• What It Will Produce
• Genomic information directly translated into
  proteins
• 10,000 to 25,000 proteins/yr
• 100,000 molecular tags/yr (initial targets)
• Biophysical and biochemical characterizations
• Research to support difficult protein and tag
  production
• Learning curve informed by successes and failures
• Research and development to import new
  technologies and methods
• Data, standards, and protocols
• What It Will Be
• 150,000-sq. ft. facility
• Advanced automated robots or production lines
• Advanced micro electro mechanical systems lab
  on a chip, microfluidics
• Synchrotron and other characterizations
• Informatics and computing infrastructure
• Cryogenic storage, handling, and shipping
• Comprehensive RD program (both at the facility
  and distributed)

Tracking proteins with tags in live cells.
Characterizing proteins with synchrotron X-rays.
32
Functions of the Facility for the Production and
Characterization of Proteins and Molecular Tags
(1) Inputs Gene Sequences

• Remote
• Characterization
• Facilities
• Specialty

• (1) Genomics
• Gene Synthesis
• Cloning
• Modification
• DNA Sequencing

• (2-3) Protein Production
• Cellular
• Cell Free
• Chemical Synthesis
• Labeling and Mutations

• Administration
• Management
• Staff Offices
• Conference
• User Offices

• (5) Affinity Reagent Production
• Libraries
• Synthesis

• (4) Characterization
• Quality Assurance
• Quality Control
• Biophysical/Biofunctional

• (8)
• Technology Research
• and Development
• High Production
• Automation
• Computing Programs

• (6)
• Computing/Information
• LIMS and workflow
• Data and Tools
• Simulation and Analysis
• Production Strategies

• (7)
• Cryogenic Archives
• Shipping
• Receiving
• Storage

• Outputs
• Proteins 2,3,7
• Affinity Reagents 5,7
• Characterizations 4
• Protocols and clones 1,3,4,5,6,7
• Data and Tools 1,2,3,4,5,6,7,8

University/Industry Technology RD Partners
33
(1) Genomics and (2) Lab-on-a-Chip Preproduction
Screening Lines
Major Equipment Layout

(1)
(2)
Incubator
Rapid Translation Systems
LC
Dispensing And Harvesting Robots
Lab-on-a-chip Nanoliter Pre-production Screening
Technologies
Light Scattering
Public and GTL Data Bases
Computing And Information Tools and Systems
Lab on a Chip Micro- Electrophoresis Separations
Lab on a Chip Expression
Dispensing Robots
DNA sequencers
DNA synthesizers
PCR machines
Colony Picker
UV Absorbance
(2)
(1)
Successful Protocols To Mass Production
Cell-free Transcription And Translation
Expression Verification
Purification By multiple Conditions
Purity and Solubility
Annotated DNA sequence
Computed Methods protocols
Verify Sequence
Amplification With PCR
Ligation/ Purification
DNA Oligonucleotides synthesized
Clones
Cellular Vectors
In Vivo Expression
Colony Picker
Transfection/ Expression
Data and All Protocols To Data Base
(6-7)
(6-7)
Materials Archived for Production
Schedule Prescreening
Validation
Compute Amino Acid Sequence
Production Chemical Synthesis
Chemical Synthesis Micro Test
Characterization
Compute Feasibility and Variants
Process Diagram

• Outputs
• gt200 successful
• protocols/day
• Preliminary Characterizations
• Genome Annotation

Gene Sequence Inputs to Protein Production (500
starts/day)
Comparative Computational Determination of
Methods and Strategies (Process terabytes of
Data/day)
Preproduction Screens Utilizing Lab-on-a-Chip
Technologies (gt100,000 trials/replicates/day)
Prepare Genes for Multiple Production
Modalities (2000/day)
Production Targets
34
(3) Protein Production and (4) Characterization
Lines
Major Equipment Layout
(3)
(4)
Production Robots
Automated Peptide Synthesis And Ligation Systems
Chemical Synthesis
Flow Systems
Analytical Robots
Dispensing And Harvesting Robots
Robot Incubator/ Shakers
Rapid Translation Systems

• Liquid
• Chromatography
• Capillary
• Electrophoresis
• Affinity Columns

Mass Spec
FTIR
UV Circular Dichroism
SAXS SANS
X-Ray Absorption Fine Structure
Cell-Free
UV Absorbance
Enzyme Activitiy
Size Exclusion Chromatograph LLS
Electron Microscopy
Fluorescence Emission Lifetime
Dispensing And Harvesting Robots
Multisample Fermentors
Automated Centrifuges
Cellular
(3)
(4)
In Vitro Insertion
Translation And Transcription
Incubation
Prepared Genes
Cell-free Synthesis
Extraction and Purification
Solubility Analysis
Biophysical Characterization
Validation QA/QC
Biofunctional Assay
Stability Assessment
Refolding Protocols
Protocols
In Vivo Vectors
Inoculate into Medium Culture
18 hour Incubation
Cellular Synthesis
Automated Data Logging LIMS Protocols and
Strategy Design Super Annotation
(6,7)
Chemical Synthesis of Peptides
Chemical Ligation
Gene Sequence
Amino Acid Labeling
Chemical Synthesis
Process Diagram
Multimodal Protein Production and
Purification (500 attempts/day) (Milligram
Quantities)
Mutants and Labeled Proteins (As Needed)
Primary, Secondary And Tertiary
Structural Characterizations (1000s/day)
Multiple BIofunctional Characterizations (10,000
s/day)
Proteins, Data, and Protocols To
Users (100s/day)
Production Targets
35
Computing Environment For All Production Lines
(5) Affinity Reagent Production Line
Major Equipment Layout
Hardware and Software
(5)
Production Robots
Distributed Computing And DataBases
Central DataBases
Central Computing
Data Management And Modeling Tools
Screening Robots

• Liquid
• Chromatography
• Capillary
• Electrophoresis
• Affinity Columns

Analysis and Assays Robots
UV Absorbance
Isothermal Calorimetry

• Displays and
• Assays
• Phage
• Ribosome
• Bacterial
• Yeast
• Two-Hybrid

Production Robots
Surface Plasmon Resonance

• Solubility
• Dye Marker
• CD
• Mass Spec

Computing and Information Processes and Products
Comparative Analyses
Annotation and Production Strategies
Protocols
Simulations/ Workflow Planning
(6,7)
(5)
Post Synthesis Biotinylation or Secondary Tagging
Production of Proteins
Extract and Purify
Reagents And Data To Archives And DataBase
LIMS
Automated Data Capture
Data reduction And Archiving
User Access
Synthesizing Proteins With Secondary Tags
Engineer tag Into Protein Gene
Synthesize
Extract and Purifiy or Use In Situ
Characterization
Production Automation
Expert Systems
Facility Models
Virtual Facility
Acquire Libraries
Amplify Libraries
Present Target Protein
Affinity Determination
Display Selection
Produce Tag In Quantity
Library Method
Data Management
Data Interpretation
Modeling
Process Refinement
Linear and Constrained Peptides
Process Diagram

• Data Archiving
• Genome Annotation
• Cloning
• Purification
• Libraries
• Affinity Selection
• And Screening
• Characterization
• Etc.

• Infrastructure
• Machine Environment
• Storage Infrastructure
• Network
• Distributed Systems

Data, Protocols, and Affinity Reagents
to Users (1000s/day)
Multiple Affinity Reagents for Each
Protein (1000s of attempts/day)
Comprehensive Data and Protocols to Production
and Archives (1000s/day)
Production Targets
36
Technology Options for Protein Production
June 14-16, 2004 GTL Technology Deep Dive
Workshop, Working Group on Genome-Based Reagents
37
Technology Options for Molecular Tag Production
June 14-16, 2004 GTL Technology Deep Dive
Workshop, Working Group on Genome-Based Reagents
38
Potential Protein Characterizations(slide being
worked)

• quality control
• assignment of possible function
• structural-activity relations
• high-throughput thermodynamic stability
• probing the folding landscape
• identification of reconstitution conditions
• computationally predict protein properties (more
  efficient production)
• discovering substrates (orphan enzymes)
• identification of co-factors (metals, NADH, ATP,
  etc.)
• identify long-term storage conditions (what keeps
  the protein from aggregating or losing activity)
• biological affect of post-translational
  modifications
• intermolecular interactions (dissociation
  constants)
• centralized and standardized characterization
  assays for computational analysis
• identification of DNA/RNA binding sites
• methodology for screening thousands of variants
  for desirable enzymatic activities
• feedback on protein production computational
  analyses
• screen small molecule natural product libraries
  for modifiers (i.e., agonists and antagonists)
• identify the ordered and disordered regions
  within a protein (provides insights into binding
  partners)

June 14-16, 2004 GTL Technology Deep Dive
Workshop, Working Group on Genome-Based Reagents
39
Elements of Facility 1 Construction Project Scope
and Cost
40
Factors in Central/Distributed and Lease/Buy
considerations

• DOE project management Order 413.3 applies
  equally to built, leased, modified
  central/distributed
• The project delivers a fully functional facility
  at CD4
• Lease Considerations (for identical facilities)
• Integrated cost analyses show cost of leased
  exceeds cost of build by year 9 after start of
  project.
• Mortgages program dollars
• All RD and equipment costs remain identical
  usually final building design follows final
  equipment acquisition decisions
• For existing buildings modification costs can be
  large
• Much of this facility is specialty space
  characterization instruments, biological
  isolation, cryogenic storage, etc.

41
Factors in Central/Distributed and Lease/Buy
considerations

• Central vs. Distributed
• End to end performance would require duplication
  of equipment, people, RD higher cost, lower
  performance
• JGI showed qualitative gains in quality,
  throughput and cost only after consolidation and
  strict focus on production goals
• Critical mass in people, facilities, learning
• Total building costs will increase if space is
  not contiguous
• Distributed functions might include
• RD and methods development
• Specialty Characterizations
• Affinity reagent and detergent libraries
• Selected Affinity reagents