1
“A heavy price was paid for molecular biology’s 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 gone……Our task is to resynthesize
biology…... The time has come for biology to enter the nonlinear
world.”
Carl Woese, MMBR 2004
The Shifting Paradigm for Biology
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”
Konopka, 2004
ASM News
70:163
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. 2:343)
Mission Science:
Requires an inherent
systems view.
3
Integrated Approach to
Shewanella
Biology
Imaging
AFM, EM, CLSM
Immuno
-
EM
Proteomics
MS
2
-
D gel
MR
-
1 Genome
Sequence,
Bioinformatics
Information Synthesis & Interpretation
Linked measurements
Concepts &
Hypotheses
Cellular networks,
Modeling
Gene Expression
Microarrays
Reporters
Physiology &
Metabolism
Computational
Biology
Data Analysis &
Integration
▲
Perturbation:
•
mutation
•
cultivation
Controlled
Cultivation
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:
(1)
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 21
st
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/docfilename/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 planet’s 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
Applications of microbial capabilities will
stimulate new biotechnology industries to put
these new processes into the marketplace.
Bioenzymes have been captured in a
ceramic nanomembrane with
improved function and stability.
Published in: J. Am. Chem.
Soc
. 2002, 124, 11242−3
Improvements in cellulase
molecular machine function
are needed to more efficiently
convert cellulose to glucose
for conversion to ethanol.
8
Subcellular location
and dynamics of
machines
Gene expression
in individual cells
Single cells
Populations
Communities
Who is expressing
what, when, where
& under what conditions?
GTL Systems Biology:
Molecules
to
Cells
to
Communities
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
1.
Investigating functions of unknown and hypothetical genes
from microbial and meta
-
genomes
The flood of genomes, unculturable systems
2.
Understanding natural systems genomic potential and
behaviors
Oceans, terrestrial, deep subsurface
Understand microbial community organization and function
3.
Investigating natural variations in critical processes and
designing and optimizing functionality
Hydrogenases, cellulases, reductases
Binding, products, biophysical parameters
4.
Enabling new single molecular investigation techniques
Tags for Protein
-
protein and protein
-
DNA interactions, e.g.
monitoring molecular structure, regulatory interactions, post
-
translational modifications
5.
Quantification standards for proteomics techniques
10
The Unknown Gene Function Problem
Hypotheticals account for >50% 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
-
gene expression (microarrays):
either a signal was detected or
not.
-
protein expression: >
500
MS/MS runs,
>
2 million
files
!
~0% FP, identifications with >90% 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%)
1/3 of Hypothetical Genes are expressed in
Shewanella oneidensis
MR
-
1
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
Topic
Oceans
(water column)
Terrestrial
(surface soils)
Deep Subsurface
(ocean, terrestrial)
Energy source(s)
& Ecologies
Primary photosynthesis
(photoautotrophs)
–
microbes at base of food
chain
Plant photosynthesis
(heterotrophs)
--
plant
-
rhizosphere
-
microbe
symbioses; Microbes are
decomposers
Reduced inorganic
compounds
(Hetero & lithoautotrophs)
--
simple communities ,
lack of predators, minimal
gene exchange
Energy &
materials storage
Rapid C & nutrient turnover,
sequestered carbon
exported
High resident carbon (plant
roots, microbes + soil
organic matter)
Resident mineralized
product, microbial biomass
& fossil organic carbon
(kerogen)
Key processes (of
relevance to DOE
missions)
Ocean Carbon Biological
Pump
--
CNPO cycles
Microbial processing of plant
organic carbon & other
nutrients; plant
-
microbe
interactions (i.e.,
mycorrhizal)
Microbial reduction of
metals, S species, nitrate;
oxidation of H2, CH4,
reduced minerals; fixation
of CO2 (autotrophy)
Mar. 3
-
4 DOE/GTL Missions Science Workshop
14
Biotech could be a significant source of
H
2
in a stabilization regime.
0
50
100
150
200
250
300
350
400
1990
2005
2020
2035
2050
2065
2080
2095
EJ/year
Electrolysis
Coal
Gas
Oil
Biomass
Biotechnology
Direct biological
production of H
2
.
Total US
End
-
Use
Energy in
2005
Jae Edmonds, JGCRI
15
BioH
2
Production
The development of methods for large
-
scale, i.e.
hundred of exajoules of energy per year
, cost
-
effective source of H
2
using biotechnology could
lower costs of addressing climate change by
trillions of dollars!
If H
2
-
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
Processes
Challenges
Deployment
Hydrogenases
Regulatory pathways
Charge transport
Partitioning
Multiple mechanisms
Integrated processing
O2 sensitivity
Range of hydrogenases
Primary and Secondary
Pathways
Electron transfer limits
Reverse reactions
Light capture
Disease Resistance
Photolytic Organisms
contained in Tubes in the
Desert
–
closed flowing
system with Hydrogen
and Oxygen separations
Photosynthetic/
hydrogen production
cassettes deployed in
Nanostructures
Research strategy:
Explore natural range of hydrogenases for variability and design principles
Explore mutations and other optimization strategies
Understand and optimize regulatory and other ancillary pathways for systems optimization
–
e.g. buildup of protons in protoplasm, alternative uses of reductants
Capture key functions for cell free incorporation into nanomembranes
Benefits:
A carbon free, renewable, secure source of energy from hydrogen utilizing fuel cells
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 (B
é
j
à
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?
Conc. H
2
SO
4
Water
Gypsum
Water
Purified
Sugar Solution
Lignin
Utilization
Ethanol
Recovery
Fermentor
Neutralization
Tank
Acid
Reconcentration
Acid/Sugar
Separation
Decrystallization
Hydrolysis
Hydrolysis
Decrystallization
Hydrolysis of Cellulose,
Hemicellulose, and Lignin
Multiple Sugar Metabolism
Alcohol Synthesis
Cellulose Today
Tomorrow?
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
19
Utilization:
1.5 Bgal Fuel Extend
Utilizing Food Starch
Marginal Cellulose
Utilization:
10 Bgal Fuel Extender
Utilizing cellulosic wastes
Large Processing Plants
Utilization:
100 + Bgal Primary Fuels utilizing
energy crops
Process Plant in a microbe
Multiple fuels
Carbon sequestration
Processes Utilized:
Starch Fermentation
Little Cellulose processing
Processes Utilized:
Acid decrystallize
–
transition to
enzymes
Cellulases
Single sugar metabolism
Multiple microbes
Some energy crops
Processes Developed:
Enzyme d
epolymerization
Cellulase, lignase, and other glycosyl
hydrolases
Transporters
Multisugar utilization
Integrated processing
Disease resistance
High temperature functioning
Designer cellulosic Energy Crops
Carbon sequestration
–
through Plant
partitioning
Biomass and Bio Fuels Benefits
Expandable and Renewable Fuel Source
–
utilize existing agriculture infrastructure
Carbon Free Fuel for Transportation
–
Carbon negative with sequestration
Carbon biosequestration in soils, Land revitalization
Compatible with fuel cell transition
–
bridge fuels to Hydrogen
Roadmap for Microbial Cellulosic Ethanol Production
Today
Interim Goals
Endpoint
20
•
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
Molecular Tags for
Single Molecule Methods
21
Single
-
Molecule Methods for the Characterization
of Protein
-
Protein Interactions and Expression
Levels in
Shewanella oneidensis
MR
-
1
Natalie R. Gassman
1*
, Achillefs N. Kapanidis
1
, Nam Ki Lee
1,&
, Ted A. Laurence
1,#
, Xiangxu Kong
1
,
and Shimon Weiss
1,2,3
1
Dept. of Chemistry and Biochemistry, UCLA,
2
Dept. of Physiology, David Geffen School of Medicine, UCLA,
3
California NanoSystems Institute,
*
Presenter:
ngassman@chem.ucla.edu
,
&
presently Seoul National University,
#
presently Lawrence Livermore National Laboratory
22
SORTING MOLECULES USING
E
,
S
D
-
only species
A
-
only species
S
(STOICHIOMETRY)
E
(FRET)
D
-
A species
0
1
0
1
D
A
D
-
A species
D
A
D
-
A species
D
A
A
D
“Fluorescence
-
aided molecule sorter”
FRET
-
efficiency ratio (
E
)
E
em
ex
em
ex ex
em
D
D
D
D D
A
F
=
F +F
(CONFORMATION INFO)
Stoichiometry ratio (
S
)
S
ex
ex
ex
A
D
D
F
=
F + F
(ASSOCIATION INFO)
FAMS
Kapanidis et al., PNAS 101, 8936 (2004)
23
Protein
-
DNA Interaction:
DNA
-
Catabolite Activator Protein (CAP) interaction
24
Protein
-
Protein Interactions of Transcription
Regulation
Two
-
component
signal
transduction,
which
regulates
transcription
by
the
alternative
sigma
factor,
s
54
,
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)
.
Enhancer
binding sites
Promoter
Transcription region
NtrC
s
RNAP
-
s
54
桯汯h湺n浥
Enhancer binding sites
RNA
-
s
54
桯汯h湺n浥
Promoter Transcription
region
s
NtrC
Complex
Promoter
Transcription region
s
Looping
intermediate to
open complex
25
Biological processes that can be dissected by
simultaneous monitoring of structure and interactions
26
Overexpression
Affinity Chromatography
Protein Production is a Bottleneck
for HT Single Molecule Methods!
Purification of NtrC
27
GTL Program
-
Microbial Community Analyses
& Cross
-
Cutting Processes
Microbial Communities
Cross cutting processes/genes
of Interest
Photolytic
H
2
generation
Contaminant
transformations
Biomass
conversion
Marine
Soil
Subsurface
Example Analyses of Mission relevant Natural
Communities and Optimization of Processes require
a minimum production of 20,000 proteins per year
28
Natural Systems
Behavior
Convert Sunlight
to Hydrogen
Convert
Cellulose
to Fuels
Toxic Metal
Reduction
Subsurface
Terrestrial
Ocean
Energy
Environmental
Restoration
•
Robust Science Base for
Policy and Engineering
•
Define Communities and
Genomic Potential
•
Understand Behaviors
•
Predict Responses and Impacts
•
Sensors
•
Genes, Proteins, Machines,
Pathways and Systems
•
Understand Behaviors
and Functions
•
Predictive Mechanistic Models
•
Systems Engineering
DOE Missions Tie to
Capabilities for Microbial Systems
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
Facility for
Production and
Characterization
of Proteins and
Molecular Tags
Facility for
Whole
Proteome
Analysis
Facility for
Modeling and
Analysis of
Cellular Systems
Facility for
Characterization
and Imaging of
Molecular Machines
Cellular Components
Providing the basis for
determining protein
function and cellular
processes
Understanding how
molecular machines are
formed and how they
function
Genomics:GTL Facilities
-
Enabling Cellular Systems Science
Developing a predictive
understanding of the
functions of cells and
communities of cells
Cellular Activities
Understanding how cells
respond to environmental
cues
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 R&D program (both at the
facility and distributed)
Tracking proteins with tags in live cells.
Characterizing proteins with
synchrotron X
-
rays.
32
(1)
Genomics:
•
Gene Synthesis
•
Cloning
•
Modification
•
DNA Sequencing
(2
-
3)
Protein Production:
•
Cellular
•
Cell Free
•
Chemical Synthesis
•
Labeling and Mutations
(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
Administration
:
•
Management
•
Staff Offices
•
Conference
•
User Offices
Remote
Characterization
•
Facilities
•
Specialty
University/Industry
Technology
R&D Partners
Functions of the Facility for the Production and
Characterization of Proteins and Molecular Tags
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
(1)
Inputs:
Gene Sequences
33
Computing
And
Information
Tools and Systems
Incubator
Rapid
Translation
Systems
DNA
sequencers
LC
DNA
synthesizers
PCR
machines
(1)
Genomics and
(2)
Lab
-
on
-
a
-
Chip Preproduction Screening Lines
Annotated
DNA
sequence
Compute
Amino Acid
Sequence
Schedule
Prescreening
Computed
Methods &
protocols
Clones
Verify
Sequence
Amplification
With PCR
Ligation/
Purification
DNA
Oligonucleotides
synthesized
Cell
-
free
Transcription
And Translation
Expression
Verification
Purification
By multiple
Conditions
Successful
Protocols
To Mass
Production
Public and
GTL Data
Bases
Production
Chemical
Synthesis
Colony
Picker
Dispensing
And
Harvesting
Robots
Cellular
Vectors
In Vivo
Expression
Colony
Picker
Transfection/
Expression
Purity
and
Solubility
Lab on a Chip
Micro
-
Electrophoresis
Separations
Lab on a
Chip
Expression
UV
Absorbance
Light
Scattering
Lab
-
on
-
a
-
chip Nanoliter Pre
-
production
Screening Technologies
Process Diagram
Data and
All Protocols
To Data Base
Chemical
Synthesis
Micro Test
Characterization
Compute
Feasibility and
Variants
Gene Sequence
Inputs to Protein
Production
(500 starts/day)
Comparative Computational
Determination of Methods and
Strategies
(Process terabytes of Data/day)
Prepare Genes
for Multiple
Production Modalities
(2000/day)
Preproduction Screens
Utilizing Lab
-
on
-
a
-
Chip
Technologies
(>100,000 trials/replicates/day)
Outputs:
•
>200 successful
protocols/day
•
Preliminary
Characterizations
•
Genome Annotation
Dispensing
Robots
Materials
Archived for
Production
Major Equipment Layout
Production Targets
Validation
(1)
(1)
(2)
(6
-
7)
(6
-
7)
(2)
34
Prepared
Genes
In Vivo
Vectors
In Vitro
Insertion
Inoculate into
Medium
Culture
18 hour
Incubation
Extraction and
Purification
Translation
And
Transcription
Incubation
Solubility
Analysis
Chemical
Synthesis of
Peptides
Chemical
Ligation
Biophysical
Characterization
Validation
QA/QC
Gene
Sequence
Biofunctional
Assay
Mass Spec
UV
Absorbance
FTIR
Enzyme
Activitiy
UV
Circular
Dichroism
Size
Exclusion
Chromatograph
LLS
SAXS
SANS
Electron
Microscopy
Dispensing
And
Harvesting
Robots
Dispensing
And
Harvesting
Robots
Multisample
Fermentors
•
Liquid
Chromatography
•
Capillary
Electrophoresis
•
Affinity Columns
Automated
Centrifuges
Robot
Incubator/
Shakers
Rapid
Translation
Systems
Cell
-
Free
Cellular
Chemical
Synthesis
Amino Acid
Labeling
Fluorescence
Emission
Lifetime
X
-
Ray
Absorption
Fine
Structure
Stability
Assessment
Refolding
Protocols
Analytical Robots
Automated Peptide Synthesis
And
Ligation Systems
Production Robots
Major Equipment Layout
Protocols
Automated Data Logging
LIMS
Protocols and Strategy Design
Super Annotation
Process Diagram
(3)
Protein Production and
(4)
Characterization Lines
Multimodal Protein
Production and
Purification
(500 attempts/day)
(Milligram Quantities)
Mutants and Labeled
Proteins
(As Needed)
Primary, Secondary
And Tertiary Structural
Characterizations
(1000’s/day)
Multiple
BIofunctional
Characterizations
(10,000’s/day)
Proteins, Data, and
Protocols To
Users
(100’s/day)
Production Targets
Flow Systems
Chemical Synthesis
Cell
-
free Synthesis
Cellular Synthesis
(3)
(4)
(4)
(3)
(6,7)
35
Production
Robots
Surface
Plasmon
Resonance
Acquire
Libraries
Amplify
Libraries
Present
Target
Protein
Linear and
Constrained
Peptides
Engineer tag
Into Protein
Gene
Post Synthesis
Biotinylation or
Secondary Tagging
Synthesize
Reagents
And Data
To Archives
And
DataBase
Screening
Robots
Extract and
Purifiy or
Use In Situ
Affinity
Determination
Display
Selection
UV
Absorbance
Isothermal
Calorimetry
•
Liquid
Chromatography
•
Capillary
Electrophoresis
•
Affinity Columns
Production of
Proteins
•
Solubility
•
Dye Marker
•
CD
•
Mass Spec
Displays and
Assays
•
Phage
•
Ribosome
•
Bacterial
•
Yeast
•
Two
-
Hybrid
Distributed
Computing
And
DataBases
Central
DataBases
Central
Computing
Major Equipment Layout
Process Diagram
(5)
Affinity Reagent Production Line
Computing Environment
For
All
Production Lines
Analysis and Assays Robots
Production Robots
Data
Management
And
Modeling
Tools
Hardware and Software
Comparative
Analyses
Annotation and
Production
Strategies
Protocols
LIMS
Automated Data
Capture
Data reduction
And
Archiving
User
Access
Production
Automation
Expert
Systems
Facility
Models
Simulations/
Workflow
Planning
Data
Management
Virtual
Facility
Data
Interpretation
Modeling
Process
Refinement
Data Archiving
•
Genome Annotation
•
Cloning
•
Purification
•
Libraries
•
Affinity Selection
And Screening
•
Characterization
•
Etc.
Infrastructure
•
Machine Environment
•
Storage Infrastructure
•
Network
•
Distributed Systems
Computing and Information
Processes and Products
Multiple Affinity
Reagents for
Each Protein
(1000’s of attempts/day)
Comprehensive Data
and Protocols to
Production and Archives
(1000’s/day)
Data, Protocols, and
Affinity Reagents to
Users
(1000’s/day)
Production Targets
Produce Tag
In Quantity
Extract and
Purify
Characterization
Synthesizing
Proteins With
Secondary Tags
Library
Method
(5)
(5)
(6,7)
36
E. coli
Cell
-
Free
Alternati
ve Hosts
Chemical
Synthesis
Homologo
us Hosts
Purification
Strengths
Established
methods, vectors
Renewable
Very cost
effective for
industrial scale
quantitties
Scalable
Ready automation
Simplified cloning
HT screening under
readily manipulated
conditions
Cofactors
Labels
Production of toxic
proteins
Some higher
success rates
for certain
proteins
Scalable
Potential for
automation
Incorporate
labels and
unusual amino
acids during
synthesis
Eliminates
codon bias or
missing
cofactor issues
Some tags
demonstrated as high
throughput, scalable
Numerous
chromatography
reagents available
Weaknesses
Scalability and
high throughput
automation
Currently only
spontaneous disulfide
bond formation
Less
developed
methods,
vectors
Cost
Not high
throughput
Ligations only at
Cys
Refolding
required
Large efforts to
develop
methods,
vectors, strains
Scalability &
high
throughput
automation
Tag removal
Tag interference
Development
Targets/Needs
More strains,
vectors, strains,
procedures for
difficult proteins
Demonstrate
automation
Directed disulfide
bond formation
Difficult proteins
Improved
vectors,
strains,
procedures
for difficult
proteins
Solve protein
folding problem
Automate for
high throughput
Generalized
procedures to
engineer
uncharacterize
d microbes
Capability to predict
effects of tags
Microfluidics
Integration with
characterization
Predictive capability
for best purification
and storage
Technology Options for Protein Production
June 14
-
16, 2004 GTL Technology Deep Dive Workshop,
Working Group on Genome
-
Based Reagents
37
Phage Display
Yeast Display
Ribosome and
puromycin display
DNA or RNA
Aptamers
Immunization of
animals
Strengths
Good diversity
Fusion
proteins
Liquid and
fluorescence
-
based screening
Affinity
maturation
Fusion proteins
Good diversity
Fusion proteins
Good diversity
Many secondary
antibodies
available
Weaknesses
Slower
screening
Plate
-
based
Required
fluores. tags
may complicate
recognition
Reduced Cys on
targets
problematic
Slower screening
Fewer
secondary
affinity labels
Not protein
based, so no
fusion proteins
Expensive
Not high
throughput
Non
-
renewable
unless use MAB
Slow
Development
Targets
Needs
Demo high
throughput
Improved
screening
High throughput
Improved
screening
Secondary
antibodies must
be developed
Not applicable
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
$-
$50.0
$100.0
$150.0
$200.0
$250.0
Facility
Equipment
Pilots, R&D,
software,
tools, etc.
Total
Contingency (25% rate)
Escalation (11.8% rate)
Prj. Mgmt. (2.7% factor)
Pilots
R&D, conc. design, startup
opns.
Software & tools dev.
Robots
Res. Equip.
Constr. Mgmt.
Design & Inspection
Facility equipment
Construction of 60K nsf labs &
184 offices (151K gsf)
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 R&D 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, R&D
–
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:
R&D and methods development
Specialty Characterizations
Affinity reagent and detergent libraries
Selected Affinity reagents
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