Presentation - The Alliance for Earth Observations

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21 Φεβ 2013 (πριν από 4 χρόνια και 4 μήνες)

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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