Genomics:GTL

Technologies for Cellular Systems

Technologies for analysis and modeling of individual cells and molecular processes within cellular systems will provide scientists with insight into the responses and functionality of microbes and microbial communities in complex environments. Modeling and real-time functional mapping of processes from the molecular through the ecosystem levels will be employed to derive a predictive molecular-level mechanistic understanding of biological function.

Scientific and Technological Rationale

Technologies for analysis and modeling of individual cells and molecular processes within cellular systems will be needed to provide the ultimate integration of analytical capabilities and knowledge synthesis critical for systems biology. These capabilities will be used to investigate how plants and microbial communities and their subsystems of cells function together to sense, respond to, and modify their environment. They will accomplish this by dynamically identifying, localizing, and quantifying molecular machines and all other important biomolecules and their interactions as they carry out their critical roles throughout plant, microbial, and community lifecycles. This grand challenge for biology ultimately must be addressed before scientists can develop and test models to predict the behavior of microbes and take advantage of their functional capabilities.

These instruments and methods will provide the ultimate testing ground for fully integrated models developed from component models created from ongoing research and from previous data, modeling, and experimentation. The experimental capabilities will drive a new generation of systems models. Essential aspects of the computational challenges and conceptual roadmaps are described in Creating an Integrated Computational Environment for Biology.

Other technologies (protein production, molecular machines, and proteomics analyses) will provide new high-throughput production and analysis capabilities to define and understand component parts and processes of microbial systems and analyze physiological and molecular conditions on a global level (i.e., measure the properties of samples comprising large numbers of cells). One of the key insights from recent research, however, is that microbial communities are dynamic and highly structured physically and functionally, suggesting that ensemble measurements that look at properties averaged across many cells can reveal only part of the picture.

To address this challenge, cellular systems technologies will focus on dynamic systems-level studies, ranging from molecular processes within individual living cells to complex, structured microbial communities or higher organisms. Microorganisms in such communities—microbial mats and biofilms, for example—occupy various microniches established as a result of coupled biological, chemical, and physical interactions. Each member of the community carries out unique functions that can vary in space and time but are integral to community stability and overall function. Cellular Systems capabilities will allow the spatial and temporal analysis of these complex microbial systems in a concerted and integrated way, from molecular processes to ecosystem functionality. This is a daunting challenge, partly because the complex multicellular drama is playing out at submicron scales. Nonetheless, we will need to dynamically image and functionally analyze the critical substructures and molecular species within microbes and their communities and develop models that describe and predict their behaviors. This capability builds on the instrumentation for intracomplex imaging to determine molecular makeup and structure and on intracellular imaging to localize machines within the cell.

The analytical and conceptual challenges of this ultimate step in systems microbiology will require unprecedented technical and computational resources and infrastructure far beyond the reach of individual investigators.

Probing Mixed Microbial Populations and Communities

In microbial communities, the complex and dynamic set of interactions that we seek to analyze is taking place in an area far smaller than the period at the end of this sentence. To understand community function, we first must be able to analyze environmental and community structure and composition at high resolution (Fig. 1). On an even-smaller scale (roughly 1/500th the size of a period), we must be able to peer inside an individual microbe in a nondestructive way to locate and continually track essential biomolecules that reveal the cell's inner workings; these biomolecules include DNA, RNAs, proteins, protein complexes, lipids, carbohydrates, and metabolites. Models then must be developed to describe key features of these biological interactions within the physicochemical environment and predict how the system will evolve structurally and functionally. Robust models are required to conceptualize these intricate systems, formulate meaningful hypotheses, manage the ensuing complexity and sheer volume of information that comes from experiments, and, ultimately, allow incorporation of the resulting science into applications.

Key information we seek about microbial-community function includes:

• Community arrangement and its physical environment
• Members and where they reside in relation to each other and their environment
• Phylogenetic, phenotypic, and functional properties of members. Do the genes in one organism regulate gene expression in another organism?
• Microbial interactions among themselves and with their environment
  • Information flow (e.g., genes, signaling molecules) within the community
  • Ecological interactions (e.g., predation, symbioses) among the various members
  • Food webs and communications
  • Excretions, secretions, and consumptions
  • Interactions of secreted components and nutrients with the environment
• Energy and element flow through communities and cells and its regulation
• Intrinsic biological (genetic) and environmental factors that control the structure, stability, and functioning of communities
• Trajectory of community evolution
  • Life strategies of each population in the community
  • Role of lateral gene-transfer processes in microbial evolution and community metagenome
  • Senescence, death, and turnover rate (consequence of death?)
  • Community resilience (biodiversity, stability)

Foundations for Community Analyses

To support these analyses, we will have a growing body of knowledge (incorporated in the GTL Knowledgebase) and capabilities from work funded by other agencies and within the GTL program. These resources will include:

• Cooperative analyses of comprehensively annotated genomes of individual microbes and the community metagenome to estimate the genetic potential of individuals and the community.
• Many critical proteins encoded in the community's genome expressed and characterized to produce a substantial body of functional characterization data incorporated into gene-annotation data sets. These data will provide significant insights into "interesting" processes that need to be pinpointed and analyzed in the context of a complete system. Since studies can be performed on the basis of sequence alone, we will have circumvented the fact that these microbes are largely unculturable.
• Ability to produce multiple affinity reagents for any produced proteins and other such biomolecules as RNA and some metabolites that can be used to locate, track, and manipulate these entities within living systems. Fusion tags can be incorporated in a variety of ways.
• Extensive measurements at the global level on bulk and ensemble samples to ascertain the phylogenetic and physiological state of member microbes or the entire community under relevant conditions. Temporal relationships will be revealed through repeated sampling and process interplay via extensive linked measurements of the transcriptome, proteome, metabolome, and other biological and physicochemical variables.
• Analysis of the structure and function of critical molecular complexes in vitro and insights to determine where and in what context they carry out their cellular functions.
• Extensive databases and exploratory tools to begin deriving underlying principles at the molecular, cellular, and community levels and the ability to begin encompassing complexities in detail as processes play out in real, nonlinear, coupled systems.
• Extensive models at molecular, cellular, and community levels to support creating and simulating hypotheses in a systems context. These models and simulations will be used to design and gain insight into experimental campaigns and protocols and provide advanced knowledge of key experimental variables that must be captured in ensuing research.

Even when all this information is at hand, unraveling how all these entities and processes act together in a continuous, concerted way—from molecular to community levels—will remain a grand challenge in accomplishing DOE mission goals. All technologies that created this body of knowledge must be specialized in innovative ways to provide the same information at a microscopic (actually nanoscopic) scale. Ideally these analytical measurements should be nondestructive and done in real time in living cells within a well-defined global and dynamic system. Understanding how these individual cells interact and function as a unit-a microbial tissue in some respects—to carry out complex processes is key to unlocking their vast potential for important applications and for achieving our science goals.

Technology Descriptions

Generally, technologies for analysis of cellular systems will combine advanced computational, analytical, and experimental instrumentation for integrated analysis of spatial and temporal variations in biological systems—how, when, and why the various system components appear, disappear, function, and remain. Imaging and other modalities will determine the state of cellular systems, from the internal makeup, structure, and dynamics of individual microbial cells to complex communities and their environments. To achieve a systems-level understanding, simulation and modeling must be coupled tightly with experiments to define and analyze complex regulatory and metabolic processes in microbial cells and communities. Compound instruments will emphasize concurrent and dynamic measurements of proteins, molecular complexes, intracellular metabolites, regulatory molecules, and gene transcripts. The aim is to establish the state of cells within populations and communities as a function of changes in physicochemical and biological conditions, emphasizing measurements at spatial and temporal resolutions appropriate to the entities being measured.

Instrumentation, Quality Control, Computing, and Support

These investigations will incorporate necessary cultivation, isolation, and analysis instrumentation—extensive environmental control and monitoring, manipulating communities in real time in various ways, and temporal and spatial resolution. The exact configuration of these instruments awaits necessary technical developments as described below.

This suite of instrumentation will be highly data intensive, providing extensive linked data sets on the dynamic behavior of microbial cells and communities. It also will be compute intensive, providing unprecedented data analysis, modeling, and simulation. In addition, it will involve new computational approaches for data storage, analysis, and use in complex models. This new knowledge will be invaluable for advancing the annotation of microbial and community genome sequence, identifying regulatory and metabolic networks in microbial systems, and understanding microbial contributions to ecosystem function.

Performance and Production Requirements

• Performance of physical (i.e., structure) as well as functional (i.e., molecular profiling) mapping
• Spatial and temporal resolution to map all levels of the functional processes within a microbial community or organisms such as plants (i.e., nanometers to millimeters and microseconds to hours)
• Performance of nondestructive measurements
• Culturing capabilities for maintenance of realistic environmental conditions and microbial communities
• Integration of modeling capabilities and supporting computing infrastructure, including data-intensive bioinformatics, compute-intensive molecular modeling, and complexity-dominated systems modeling

DOE has an extensive and successful history of developing and applying new technologies to complex problems in the physical and chemical sciences. As in the genome projects, the agency can draw on multidisciplinary teams of biological, physical, computational, and other scientists and engineers from national laboratories, academia, and industry. GTL brings a tremendous opportunity for using these same talents to devise technological solutions to some of biology's most complex problems.

Technology Development for Cultivation of Microbial Communities

The classic definition of an unculturable microbe is that it cannot be grown in homogeneous suspension. Because of this, there is a dearth of information about the metabolic capacity of microorganisms that resist cultivation under laboratory conditions (Keller and Zengler 2004). This is due primarily to the difficulty or impossibility of simulating the chemistry and interspecies interactions of highly structured communities by suspended cell-culture techniques. Most microbes reside in these structured communities and display unique phenotypic states in response to microenvironments within the communities. Many current technologies require large populations of cells to measure gene expression, proteome, and metabolites, masking the true cell-to-cell heterogeneity. The inability to cultivate most structured communities formed by natural microbial populations limits our discovery of new genes, gene products, and resultant functionalities. New cultivation techniques must support the development of meaningful community structures, and the functions of community members must be measurable in that environment (see sidebar, Laboratory Cultivation Techniques to Simulate Natural Community Structure).

Cultivation systems must allow for the precise control and manipulation of environmental conditions and for the monitoring and culturing of microorganisms in meaningful community structures. Many microorganisms of scientific and biotechnological importance, including those sequenced by the Biological and Environmental Research Program, by GTL, and by the Joint Genome Institute's Community Sequencing Program, are relevant to various DOE missions. Some thrive in unusual or extreme environments or those in which gradients or temporal changes occur in physicochemical conditions.

Scientific investigations of these organisms and the communities in which they live thus require flexible, highly controlled, and instrumented systems that can provide a range of environmental conditions and sophisticated measurements. These conditions include monoculture or mixed cultures; nutrient status; extremes of pH, temperature, and salinity; exposure to contaminants and radiation; gas composition and pressure; light intensity; and the presence of solid phases. The ability to control and monitor the environment allows for rigorous investigations and interpretations of gene expression, regulation, and function at the level of individual cells, cell populations, and mixed communities. When required, cells of unusual or difficult-to culture microorganisms will be produced in sufficient quantities to provide protein for biophysical, structural, and functional analyses. In other cases, very small numbers of unusual or difficult-to-culture microorganisms might be studied using novel microscale approaches combined with specialized sensitive analyses of gene expression, proteomics, metabolism, and metabolite flux. Ultimately, for meaningful analysis of communities, measurements at the individual cell level will be essential. To select for study any cell in such a structured community, we need to be able to (1) remove it from its environment without inducing significant changes in the properties being measured or (2) conduct analyses of living cells in situ (i.e., without disrupting its environment or harming the cell).

Requirement Examples

• Development of techniques to isolate single cells from a natural community and analyze them for the expression of targeted genes, proteins, and other products (e.g., using fluorescent tags produced from metagenome sequences).
• Evaluation of metabolic processes carried out by cells in structured microbial communities, using molecular tags for RNA, proteins, or other reported moieties to map individual cell populations within the community (see imaging discussion below).
• For unsequenced microbes, application of techniques such as intact biofilm polymerase chain reaction (IB-PCR) to construct 16S rRNA clone libraries once a community is formed. The rRNA sequences of each population present could be used to establish phylogenetic links to other known populations. Techniques such as fluorescence in situ hybridization (FISH) then could employ 16S rRNA sequence data to construct oligonucleotide probes to locate different populations within the biofilm and identify putative associations among colocalized populations.
• For many systems, capabilities to assay the distribution of properties among a population of extracted cells from structured communities, sediments, or soils. Providing invaluable information, flow cytometry will be a high-throughput method of choice. Novel cultivation approaches also might be combined with single-cell sorting techniques such as emerging microfluidic lab-on-a-chip devices to grow and study currently uncultivable members of microbial communities.

Development of Genomic Capabilities

To test hypotheses about function, genetic manipulation to generate mutants and specific constructs containing tags or reporter molecules is an essential requirement for systems biology research. Highly roboticized capabilities will be essential for high-throughput construction and screening at the genome level. Examples of required basic capabilities include nucleic acid isolation and analysis, sequencing and annotation, expression analysis, gene cloning and expression, fusion tagging of genes, general tools to manipulate members genetically, and cell sorting. Molecular microbiology and, in fact, all microbiology support capabilities will require a highly developed system for information management and integration.

Technology Development for Imaging and Spectroscopy

Cellular systems analysis will employ a broad range of imaging modalities to monitor the structure of microbial communities and image (spatially and temporally resolving) the many molecular species critical to community function. While molecular machines technologies focus on imaging intracomplex structure and cellular location, cellular systems technologies focus on spatially and temporally mapping multiple processes through the lifecycle of a community of cells in a complex environment. Imaging modalities available for both applications are presented in Table 1. Characteristics of Available Imaging Modalities, Table 2. Attributes of Available Techniques for Cellular Systems Characterization, and Fig. 1, all of which list the primary probe methods available, techniques based on them, analytical characteristics, prospects for further development, and computing requirements. To be applicable, these methods must be chemically specific, perform measurements nondestructively, and be capable of functioning in concert with other techniques.

Analytical Characterization of Cellular Systems

Critical to addressing key scientific questions will be the novel application of existing and emerging technologies that characterize systems in a continuous and spatially resolved way. Analyses now conducted on bulk samples must transition to nondestructive processes capable of characterizing systems ranging from a microbial community through multiple processes within a single living cell. Also, the power to view multiple systems with high spatial and temporal resolution must be augmented with the ability to identify, track, and manipulate living microbes in the presence of other strongly interacting species. To achieve this, many classic imaging techniques must be coupled with methods that can detect specific molecules or processes. Physical, chemical, and biological variables must be identified and tracked. Furthermore, the power to observe systems in action will need to be enhanced by the ability to interact with these systems.

Developing the capability to view biological systems in great detail will enable new high-throughput approaches to studying cellular systems. Each cell in a tissue, culture, consortium, or community presents a unique reflection of the biological response to the overall system's changing state. Each provides a set of multilevel outputs in response to the effects of changing parameters (e.g., environmental insults, nutrient gradients, and temperature). To the extent that this parallel data stream can be captured in real time, biological experiments can be conducted in a high-throughput manner rather than running as several series of experiments to evaluate each possible response (e.g., cell division, movement, and protein shedding) for each type of environmental change.

To enable these advances, new technologies must address the special requirements for observing biological systems. Ideally, techniques will be nondestructive, noninterfering, and compatible with the analysis of heterogeneous, living systems. They will need to document the state of each cell (or many cells or cell types) as time and environmental conditions change. Furthermore, physical and chemical information must be mapped onto community structure while detailing changes at the molecular scale. These analyses will necessitate the development of new software to provide intelligent processing of data. Ultimately, such tools will help elucidate the large-scale biochemical organization that characterizes community structure. Such new analytical approaches will be essential for assessing the community's physiological and phylogenetic makeup and for testing predictions derived from theoretical models.

A number of these scientific needs will require fundamental new developments in imaging technology—a transformational goal for GTL biology. Revolutionary advances will be essential for determining the dynamics of communities and their functions under various environmental conditions, defining the physical structure of cells and communities, detecting and tracking extracellular and intercellular molecules to define cell states, and, ultimately, understanding how molecular events are communicated in space and time.

Examples of Analytical Requirements

Intracellular Structure. Intracellular protein, RNA, and metabolite localization and kinetics of localization.

• Proteomics on replicate communities.
• Fine-scale cell ultrastructure.
• New multimodal capabilities for dynamic imaging of targeted intracellular molecules and their interactions (including machines) in individual cells and cell assemblies [e.g., with antibody labeling and electron microscopy (EM)].

Community Structure. Analytical instrumentation and techniques for determining overall community structure and identifying and characterizing spatial and temporal variations in metabolites, signaling and regulatory molecules, and the physicochemical environment within communities.

• Probes for the in situ measurement of extracellular metabolites in real time.
• Imaging and spectroscopy of population structure, gene expression, and metabolites in cell aggregates and subpopulations within communities.
• Characterization of cells in mixed communities by multispectral imaging of key cellular chromophores, possibly moving to on-the-fly cell-sorting platforms.
• Measurement of elemental distribution, oxidation state of elements, and biomolecules within and among communities.
• Quantitative imaging of metabolite (and signaling-molecule) flux between cells in close proximity to or in contact with each other—one of the most critical needs for understanding how microbial communities function.
  • Analysis and assessment of the makeup and role of extracellular polymers in community structure, function, and stability.
  • Detection and frequency of genetic exchange, recombination, and evolution within communities.
  • Determination of macroscopic transport of water, solutes, and macromolecules and their relationship to microbial function.
• Characterization of interface physical and chemical properties.

Identified Development Needs

• Advanced chemical and biological probes, including engineered microorganisms, tagged biomolecules, and chemical sentinels that will help characterize microbial communities.
• Advanced tools for imaging characterization in the laboratory as well as in the field.

A variety of imaging and microspectroscopic techniques are emerging to meet these challenges. In general, imaging relates spatially dependent information. Characterization of additional dimensions, however, will be essential for relating system activity. Some commonly used imaging techniques include:

• Short-Wavelength Techniques. Analyses with electrons and X rays typically provide the highest spatial resolution. Although commonly associated with ultrastructural analyses, short-wavelength techniques are being extended for analyses of whole cells at atmospheric pressures. Additionally, X rays are useful for mapping trace metals, while spectroscopic measurements can provide chemical identification.
• Optical Microscopies. The current standard for live-cell imaging, these tools are ideal for studying dynamics across a broad range of time scales and are sensitive down to the single-molecule level. A number of physical scales can be assessed, and emerging techniques and new labels are improving the sensitivity and resolution of optical microscopy.
• Long-Wavelength Techniques. A variety of these procedures including vibration, magnetic resonance, and terahertz-based imaging can provide essential information on chemical structure, identity, and spatial arrangement. For example, vibrational signatures are molecularly specific and can produce direct chemical information without additional labels.
• Other Techniques. A broad range of unconventional imaging approaches are making an impact on biological studies. Most notable, the family of instruments comprising scanning probe microscopy enables molecular-scale resolution; and chemical, electrical, and physical properties can be measured simultaneously. Emerging tools based on optical and magnetic trapping are allowing measurement of mechanical properties while micro- and nanoscale structures permit sensing of chemical, physical, and biological attributes.

Clearly, many current imaging and microspectroscopic techniques possess significant attributes and provide information relevant to the study of biological systems. Significant advances still are needed to adapt many of these tools to the characterization of microbial cellular systems much smaller than eukaryotic cells. Advanced instrumentation, improved biocompatibility, new approaches for targeting and delivery of tags, and improved labels are but a few of the significant challenges that face imaging technologies. More significant, no technique alone can provide the broad range of information needed to understand community structure and system function. A combination of methods will be essential to extend the depth of information required.

Monitoring and Interacting with Cellular Systems

To enable effective systems-level studies, the ability to monitor systems in action must be enhanced with selective construction, manipulation, and interaction with the system. Only then can efficient experimental evaluations and effective iterations be achieved with pursuits in theory, modeling, and simulation. This integration will be essential for studying microbes, consortia, and microbial communities.

Advanced cultivation systems that allow for precise control, manipulation, and monitoring of environmental conditions must be compatible with advanced imaging technologies. Chemical gradients will need to be controlled and monitored precisely while temporally measuring molecular-scale properties. Genetically defined organisms must be carefully arranged into ordered microbial communities, perhaps through molecular-scale patterning techniques resulting from nanotechnologies. Such highly defined systems will require integration with sensing capabilities and the ability to activate biomolecular networks remotely. The capacity for simultaneously imaging and specifically targeting reagent release or activation, as currently used in biomedical applications, is within reach for GTL systems biology studies. The creation of such compound, multifunctional instruments will enable the collection of information needed to understand and exploit complex biological systems.

Technology Development Progress and Benefits

Advanced Optical Methods - Laser or Synchrotron Based

• Optical spectroscopic methods can be used as tools for noninvasive characterization and monitoring of dynamic behavior.
• Measurements of absorption and in vivo fluorescence can be used to monitor the presence and relative concentration of optically active biochemical species.
• Light-scattering spectroscopy can probe the size distribution of community structures.
• Vibrational (infrared and Raman) spectroscopy is a technique for studying the composition of biological materials without perturbing or labeling the sample. Biological components (e.g., lipids, proteins, nucleic acids, and carbohydrates) and biofilm and microbial surfaces (e.g., minerals and polymers) have unique vibrational spectra based on their chemical structures.
• Use of these methods will provide new information on the following:
  • Large-scale (1- to 10-micron) biochemical organization.
  • Composition and distribution of extracellular polymer matrices.
  • Concentration and distribution of nutrients, metabolites, signaling molecules, and other macromolecules.
  • Interactions of biofilms and microbial communities with supporting surfaces.

Because vibrational spectromicroscopy is noninvasive, it can be performed on dynamic living systems in combination with other techniques. If synchrotron radiation is used as the photon source, a dynamic system can be studied directly on surfaces of geological materials.

Significant progress already has been made using confocal and two-photon fluorescence microscopy. The specificity of these techniques is provided by the exogenous chromophore targeted through an affinity reagent or fusion tag to a particular protein. The resolution is on the order of a micron and slightly higher for two-photon than for confocal microscopy. Delivering chromophores to remote regions within a community or cell is a particular challenge. Additionally, the identification of probes that maintain activity in diverse environments is required.

All these techniques can be used in an imaging arrangement to monitor changes in community behavior in real time. Improvements are needed in such areas as spatial resolution, the ability to provide quantitative information, and data-acquisition speed. Additionally, advanced light-microscopy techniques can be developed for high-resolution 2D and 3D mapping. Often with specificity to particular components associated with imaging, these techniques include surface-plasmon resonance, surface-enhanced Raman spectroscopy, imaging of second-harmonic generation, optical-coherence tomography, and coherent anti-Stokes Raman scattering.

Imaging Macromolecular Complexes

Many types of imaging technologies can be employed to identify and spatially and temporally localize macromolecular complexes and their interactions within a dynamic community environment. Some specialized techniques have specific applications to the analysis of macromolecular complexes in situ in live, fixed, or frozen cells or ex situ. The strengths of imaging techniques typically include detection sensitivity and the ability to identify complexes in cells. Imaging techniques are applicable to all classes of complexes. In many cases, however, the identities of one or more components of the complex must be known to prepare tagged probes for imaging analysis. This requirement limits the application of imaging to full identification of protein complexes. Currently, most imaging techniques are relatively slow; automation, however, is providing faster sample throughput, and improved computational tools are enhancing data acquisition and analysis. Imaging techniques relevant to identification and characterization of protein complexes are summarized below, with additional information on other imaging tools in Table 2.

Tagged Localization. Used with visible, X-ray, or electron microscopies to identify sets of biomolecules labeled with tags. An in situ method applicable to live (visible light), fixed, or frozen cells, it also is applicable to tagged transient complexes and membrane-associated complexes. A limitation is that the complex must be labeled with a tag, requiring tag synthesis and introduction into cells. Spatial resolution in these modalities comes from the instrument response function of the exciting source (i.e., the exciting beam provides the resolution). More developed X-ray optics, more versatile excitation sources, and improved probes are needed. Lanthanide dyes, quantum dots, nanoparticles, tetracystein-based ligands, and other probes are examples of some recently reported probes used with various imaging modalities.

Fluorescence Resonance Energy Transfer (FRET). Used to identify pairs of biomolecules labeled with tags as well as to provide information on biomolecule relationships. This in situ method is applicable to live cells, tagged transient species, and membrane-associated complexes. FRET is particularly good for structure and binding of extracellular ligands. Like other imaging techniques, it requires tag synthesis and introduction into cells.

Scanning Probe Microscopy. Identifies protein associations by scanning with a specific molecule attached to the tip, including transient molecules. The technique is capable of very high spatial resolution (depending on the length of probe time) and of single-molecule detection. It is most suited for the study of membrane-associated complexes with whole cells or for the study of isolated complexes. Like other imaging techniques, it requires that the identity of one component of the complex be known so a molecule can be attached to the tip as the probe molecule. The probe, for example, then can be used to identify interaction sites on a cell surface. The technique is labor intensive and slow. Identification is a one-at-a-time process unless multiprobe devices with individual probe molecules are employed. These multiprobe devices are under development to allow technique application in a highly parallel fashion. Computer modeling of protein folds would enhance data interpretation, and improved computation is needed for data visualization and manipulation.

Development Options

As previously mentioned, many needed techniques have yet to be developed sufficiently to analyze microbes of less than one micron in complex and changing communities. Many potential options must be explored over the next few years to determine probe and detection modalities capable of providing necessary information under these demanding conditions. Options that may be explored regarding available techniques, their range of applicability, and information they might provide are shown in Table 2 and Table 3. Cellular Systems Analysis Technology Development Roadmap. The bulk of intracomplex characterization of molecular machines will be carried out utilizing molecular machines technologies. The sidebar, The Super Imager, details creation of super imagers comprising compound, multifunctional instruments that individually would include many of the capabilities listed.

Development of Computing Capabilities

Computational tools and infrastructure are required for efficiently collecting, analyzing, visualizing, and integrating large data sets to elucidate gene function and to model and simulate regulatory and metabolic networks, cells, communities, and ecosystems. These tools will support the development and validation of theories and models of community growth, function, and environmental response. New theory, algorithms, and implementation on high-performance computer architectures also are needed for modeling and simulating cellular systems. Enabling a broad range of biologists to access the large data sets and computational resources for discovery-based biology will require the development of web- and grid-based technologies (see Table 4. Computing Roadmap).

This Webpage adapted from Genomics:GTL Roadmap, DOE/SC-0090, October 2005. See References PDF.