Technology for Ultrahigh-Resolution Localization of Gene Transcription Regulator Binding Sites on Whole Bacterial Genomes Shimon Weiss, University of California at Los Angeles
Collaborators: Pacific Northwest National Laboratory
Summary A novel technique for identifying the targets of DNA binding proteins will be developed using quantum dots to uniquely tag both the DNA and the regulator proteins. The quantum dots can be tuned to give off specific colors. Ultrahigh resolution fluorescence microscopy will be used to image the colors and identify exactly where on the DNA the regulator proteins have bound.
Develop a Hybrid Electron Cryo-Tomography Scheme for High Throughput Protein Mapping In Whole Bacteria Huilin Li, Brookhaven National Laboratory
Cryo-electron microscopy (cryoEM) and scanning transmission electron microscopy (STEM) will be combined to locate specific proteins in the crowded environment inside microbial cells. CryoEM resolves structural features down to 10 nanometers while STEM can locate heavy metal cluster labels for specific proteins. The combination will be used to map the location of heavy metal resistance protein complexes in bacterial cells.
Single Cell Imaging of Macromolecular Dynamics in a Cell
James H.D. Cate, Lawrence Berkeley National Laboratory
The goal is visualization of specific structures within single cells. The novel approach is to genetically engineer target the structures to bind a probe and/or companion material that will fluorese upon binding. Imaging of ribosomal interactions will be the first trial arena.
Microscopes of Molecular Machines (M3): Structural Dynamics of Gene Regulations in Bacteria Carlos Bustamante, Lawrence Berkeley National Laboratory
Collaborators: Pennsylvania State University
Powerful and complementary microscope technologies will be developed to study the structure and changes that occur when multi-protein molecular machines are formed. The four technologies that will be used, cryo-electron microscopy, atomic force microscopy, optical tweezers and single-molecule fluorescence microscopy, look at molecular machines from different but complementary perspectives. Cryo-electron microscopy can, for example, form visual images of an entire molecular complex, while single-molecule Fluorescence can show real time formation of complexes as a result of fluorescence signals that can be seen as specific proteins come in contact with each other.
Real-time Expression Profiling of Single Live Cells of Shewanella oneidensis Xiaoliang Sunney Xie, Harvard University
Collaborators: Pacific Northwest National Laboratory
The capability to make real-time observations of gene expression in live cells of the microbe Shewanella oneidensis with high sensitivity and high throughput will be developed. The research will demonstrate new fluorescent probes that can be used to measure the full range of proteins in a cell, from proteins that have only a few copies to those that have many copies. A microarray of identical living cells will be created, each cell of the array will be modified to produce a fluorescent signal for only one of the cell’s many proteins. Scanning the entire array for signals from each cell can reveal changes in the level of specific proteins over time, under the same or varying environmental conditions.
Probing Single Microbial Proteins and Multi-proteins Complexes with Bioconjugated Quantum Dots Gang Bao, Georgia Tech Research Corporation
Collaborators: Emory University, Carnegie Mellon University, California Institute of Technology
A quantum dot-based strategy will be developed for identifying and imaging the location and movement of individual proteins and protein-complexes in microbial cells. Quantum dots, are nanometer sized crystals that can emit a very specific color depending on their size. Specific dots will be attached to small biological molecules (ligands) that can enter the cell and stick to specific proteins or protein complexes. The location of the protein or complex can then be determined using optical imaging and electron microscopy.
Development of Advanced Tools for Data Management, Integration, Analysis and Visualization Through a Comprehensive Systems Analysis of the Halophilic Archaeon Leroy Hood, Institute for Systems Biology
Collaborators: Keck Institute
Partial funding is being provided to develop and provide tools that will be useful for biologists for a) data integration, b) network inference, c) database mining, and d) algorithms to compare networks.
Rapid Reverse Engineering of Genetic Networks via Systematic Transcriptional Perturbations J.J. Collins, Boston University
A novel procedure called “real competitive PCR (rcPCR)” will provide proteomics data that will be used to reconstruct microbial regulatory networks. The team has tested the lab work and modeling on a simple E. coli system and propose to expand it to Shewanella.
Computational Hypothesis Testing: Integrating Heterogeneous Data and Large-Scale Simulation to Generate Pathway Hypotheses Mike Shuler, Gene Network Sciences (connection to Cornell University)
Collaborators: Wadsworth Center, Washington U, St. Louis, Cybercell consortium
A dynamic simulation of E. coli and Shewanella will be developed using published literature, databases, and high-throughput E. coli experimental data from the Cybercell consortium, including microarray, proteomics, and quantitative RT-PCR. The PIs will start on a hypothetical network, move to E. coli, and extend their simulations to Shewanella. Relevant software will be made readily available. The investigators will form a strong connection with the Shewanella Federation.
Computational Resources for GTL Herbert M. Sauro, Keck Graduate Institute; Claremont College
Biospice is a significantly used, open-source software system funded primarily by DARPA. The PI will enhance the Biospice software consistent with GTL priorities in networks and modeling. This will enable GTL to support open-source software and be part of an ongoing community modeling effort.
Development of Bioinformatics and Experimental Technologies for Identification of Prokaryotic Regulatory Networks Charles E. Lawrence, Wadsworth Center Collaborators: Washington University, St. Louis
Shewenella transcription factors and transcription factor binding sites will be predicted mathematically and those predictions will be tested experimentally. The PGF will determine the genomic sequence of additional strains of Shewenella providing information essential for this type of comparative modeling approach.
Microbial Genomics and Metabolomics
Growing Uncultured Microorganisms from Soil Communities Kim Lewis, Northeastern University
Groundwater and soil communities associated with contamination by metal and uranium will be characterized by 16S rRNA analysis and using various approaches for culturing of the microbial communities. A simulated in situ environment developed by the PI will be used in addition to the use of “helper” organisms and the adaptation of some of the same organisms. Whole genome sequencing or comparative genome hybridization studies will be used to assess diversity and metabolic properties of some of these organisms.
Application of High-Throughput Gel Microdroplet Culturing to Develop a Novel Genomics Technology Platform Martin Keller, Diversa Corp
Collaborators: LANL, PNNL
This project will develop and improve on three areas of prime importance to environmental microbial genomics using a gel microdroplet culture technique. Specific microbes of interest will be identified microdroplets using rRNA probes or fluorescent probes for functional genes of interest. Complete genomic DNA will be amplified from microbes in single microdroplets, providing a template for shotgun library generation and whole genome sequencing. Finally, mRNA will be amplified from small numbers of cells and the sensitivity of detection methods for use in microarrays will be improved.
Technology for Combined High-Throughput Proteomic and Metabolomic Studies of Microbial Communities Richard Smith, Pacific Northwest National Laboratory
Collaborators: University of Massachusetts at Amherst
Accurate mass tag (AMT) technology will be used to study the proteome/metabolome of microbial communities. The project will initially focus on Geobacter-dominated communities. Stable isotope labeling technology will be used to monitor the metabolism and physiology of a microbial community.
Metabolomic Functional Analysis of Bacterial Genomes Cliff Unkefer, Los Alamos National Laboratory
Collaborators: Oregon State University
The “metabolome” of the ammonium-oxidizing bacterium Nitrosomonas europaea will be examined. Mass spectrometry or nuclear magnetic resonance will be used to analyze metabolites produced by wild type or mutant cells that were fed stable-isotope labeled compounds. The use of labeled compounds will enable identification of not just the concentrations but also the pathways and fluxes of metabolites involved in essential metabolism.
New, Highly Specific Vibrational Probes for Monitoring Metabolic Activity in Microbes and Microbial Communities Thomas Huser, Lawrence Livermore National Laboratory
Specific probes will be developed consisting of nano-scale particles that yield specific Raman signals (e.g. vibrations) when the particle is in contact with targeted intercellular or intracellular compounds. These nanoparticles will act as localized sensors to measure metabolites in microbial communities and within individual microbes. A goal is to develop nanoparticles small enough to study intracellular metabolite concentration and heterogeneity. Initial targets will be pH, glucose, and oxygen.
New Technologies for Metabolomics Jay Keasling, Lawrence Berkeley National Laboratory
Collaborators: Pacific Northwest National Laboratory, University of Massachusetts at Amherst
New methods for the comparative profiling metabolites in microorganisms before and after perturbation will be developed for Shewanella oneidensis and Geobacter metallireducens. Metabolic fluxes with respect to the perturbed metabolism will be measured. Nuclear magnetic resonance will be used to study flux through metabolic pathways since this technology allows for the detection of relatively small amounts of metabolites.
Protein Production and Molecular Tags
Development of Multipurpose tags and Affinity reagents for Rapid isolation and Visualization of Protein Complexes Thomas Squier, Pacific Northwest National Laboratory
Small fluorescent molecules (probes) and proteins tags will be developed that can be used to detect, isolate, and cross-link proteins and multi-protein complexes. The fluorescent molecules will be used to visualize the distribution of specific proteins or complexes in a living cell. The chemical modification of the fluorescent molecules will be used to link proteins that form complexes allowing the identification of which molecules work together as key molecular machines.
A Combined Informatics and Experimental Strategy for Improving Protein Expression John Moult, University of Maryland Biotechnology Institute
The proposed research seeks to understand the reasons why a significant percentage, perhaps, 50% of non-membrane proteins, are not soluble and therefore cannot produce crystals needed for determining highly detailed structures using techniques such as x-ray crystallography. The research will examine the reasons for differences between successfully produced protein and the rest by comparing a variety of biophysical and biological characteristics of proteins such as thermodynamic stability, protein folding behavior. The technology of micro arrays that can measure increases or decreases of the abundance of proteins in the cell will be used to determine if specific proteins or groups of proteins influence the success or failure of producing soluble molecules. This information will be compiled made available on a public database.
Development of Genome-Scale Expression Methods Frank Collart, Argonne National Laboratory
Collaborator: Roche Diagnostics
A high throughput protein production strategy will be developed for difficult to produce cellular proteins, including proteins in the cell membrane, in the periplasm (space between the outer cell wall and inner cell membrane) and high molecular weight and insoluble proteins. The studies will use two important microbes Shewenalla oneidensis and Geobacter sulfurreducens and examine the usefulness of techniques for protein production in living cells and outside the cell (cell free). The result of this work would be to make available methods that will assist researchers in predicting successful strategies for protein production.
High-Throughput Biophysical Analyses of Purified Proteins William Studier, Brookhaven National Laboratory
This project will develop and test techniques and biological resources to be used in a high throughput system for protein production. A complementary activity is proposed to develop a prototype high throughput system using technologies based at the National Synchrotron Light Source (NSLS) to rapidly determine some basic protein characteristics such as size, shape, types of bound metals. The four techniques, small angle x-ray scattering, ultra violet spectroscopy, infrared spectroscopy and x-ray absorbance micro-spectroscopy if successful would take advantage of the high intensity beams of NSLS to produce rapid analyses.
Chemical Methods for the Production of Proteins Stephen Kent, University of Chicago
A simple chemical method using low cost hardware will be developed to improve the chemical synthesis of protein that are difficult to produce using biological based systems such as those employing recombinant DNA methods. Small proteins and integral membrane proteins that are difficult to make using current methods are the primary targets of this effort. The proposed approach will focus on generating the needed milligram quantities of these protein based on their peptide composition predicted from gene sequence data.
Fluorobodies: Binding Ligands with Intrinsic Fluorescence Andrew Bradbury, Los Alamos National Laboratory
A method for high throughput production of molecules called fluorobodies will be developed. Fluorobodies can binding with and signal the location of specific proteins of interest. Fluorobodies are molecules made by grafting protein binding loops from antibodies, called Complimentarity Determining Regions (CDR), that strongly attach to specific proteins, onto a biological molecule called a Green Fluorescent Protein (GFP) that emits a strong fluorescent signal. The combination can be used to easily detected, identify, measure and isolate specific proteins.
Ethical, Legal, and Societal Issues
Science Literacy Workshops Barinetta Scott, SoundVision Productions
This project will convene a series of workshops for public radio science journalists focused on post-genomic science, including core concepts of the Genomics:GTL Program.
The DNA Files Barinetta Scott, SoundVision Productions
This project extends an earlier project developing radio presentations about genomic science to create an hour-long public radio documentary on the Genomics:GTL Program. The goal is to teach the basics of systems biology and help listeners contemplate the social, ethical, and legal issues emerging from the genome projects.
Unless otherwise noted, publications and webpages on this site were created for the U.S. Department of Energy Genomic Science program by Biological and Environmental Research Information System (BERIS). Permission to use these documents is not needed, but please credit the U.S. Department of Energy Genomic Science program and provide the URL (https://genomicscience.energy.gov). Materials provided by third parties are identified as such and not available for free use.
Unless otherwise noted, publications and webpages on this site were created for the U.S. Department of Energy Genomic Science program by Biological and Environmental Research Information System (BERIS). Permission to use these documents is not needed, but credit the U.S. Department of Energy Genomic Science program and provide the URL https://genomicscience.energy.gov. Materials provided by third parties are identified as such and not available for free use.