Genomics:GTL

Technologies for Cultivation of Microbial Communities

Precise control, manipulation, and monitoring of environmental conditions; interrogation

Functional individual microbial cells in the context of characterized physiochemical environment

Support for formation of structured communities

Requirements defined for analyzing individual cells within structured communities:

Mixed microbial cultures

• Suspended and structured
• Biofilms

Methods and approach to identify and track microbes and molecular complexes

• Tagged probes
  • Increased variety of signals
  • Signal interpretation
  • Incorporation in cell
• Arrays

Multiple flexible experimental systems to control and manipulate growth and conditions with multiplex measurements of activity including:

• Chemostats
• Microtechnologies
• Remote sensing
• Imaging
• Surfaces to nucleate biofilms and other structures

Multiple probes to identify community members

Temporal monitoring of community structure and function

Integration of culturing capabilities within multiprobe instrumentation for simultaneous control, manipulation, and multimodal analyses of structured communities:

• Nondestructive
• Real time
• Linked databases
• Environmental, community, cellular, and molecular levels

Integrated, highly characterized, and real-time manipulatable structured microbial communities that simulate natural communities and niches:

• Protocols
• Extracted samples
• Characterizations
• Analytical images

High-Throughput Cultivation for Single-Cell Analysis

Sampling techniques

Controlled viable growth of single cells

Analysis from within structured communities, in microculture extracts, or in place:

• Cell sorters
• Lab on a chip and microfluidics
• Single-cell analysis of "unculturable" environmental samples

Assessment of compatibility with analytical instrumentation and simulation fidelity of natural environments

High-throughput operational mode combining culturing techniques interfaced with multimodal, analytical, and manipulation modalities

Single cells prepared in conditions that simulate microniche environments in highly structured microbial communities such as biofilms (formerly unculturable)

Temporal and Spatial Localization of RNAs, Machines, and Metabolites

Analytical measurement contexts:

• Environmental
• Community
• Intercellular
• Intracellular

Requirements defined for multimodal measurements:

• Environmental physicochemical variables
• Intercellular biomolecules
• Intracellular biomolecules
• Metabolites
• Community overall biochemical and biophysical functionality

Examples of needed instrumentation with molecular specificity, sensitivity, and spatial and temporal resolution:

• NMR for community-scale microscopy (e.g., metabolites, signaling molecules)
• Small molecules in living cells
• Gene expression in living cells
• Proteins and machines in living cells, including dynamics and interactions
• Biomolecular mapping microscopies [confocal, CryoEM, SPM (AFM, STM, others)]
• Image-interpretation tools
• Visualization
• Computational systems
• Databases

Modular analytical and imaging instrumentation and methods integrated with culturing, monitoring, control, and manipulation modalities to assess:

• Viability of integrated approaches
• Compatability with living systems
• Intermodal interactions
• Ability to meaningfully assess single cells
• Data integration
• Simulation and modeling integrated into experimental methods
• Visualization of multimodal analyses and system monitoring and manipulation

Integrated culturing capabilities within multiprobe instrumentation for simultaneous control, manipulation, and multimodal analyses of structured communities:

• Nondestructive
• Real time
• Linked databases

Characterizations of microbial communities in realistic environments at the environmental, community, cellular, and molecular levels:

• Spatial
• Temporal
• Functional
• Process
• Molecular

Databases and query tools

Protocols

QA/QC