Facilities and Resources
Oak Ridge National Laboratory
The Plant Metabolomics and Physiology facilities have a well-equipped physiology/biochemistry lab that is instrumented for metabolite analyses. The lab has a ThermoElectron TEMPUS Fast Gas Chromatograph Time-of-Flight Mass Spectrometer that is configured for both electron impact ionization (EI) and chemical ionization (CI), a Hewlett-Packard HP5980/5972 quadrupole gas chromatograph/mass spectrometer configured in EI mode, an HP1090L high performance liquid chromatograph with a built-in diode array detector, an HP1046A fluorescence detector, and an HP1049A electrochemical detector. There are two -80°C deep freezers for sample storage, several rotary evaporators, a freeze drier, and a Speedvac for sample preparation. Additionally, there is a Carlo-Erba NA1500 analyzer for in-house determination of C and N concentrations of plant tissues, and spectrophotometers for chlorophyll determination. Several LiCor 6400 open-flow gas exchange systems are available for photosynthesis measurements.
The Genomics facilities are outfitted with all the equipment necessary to perform the described research. Standard equipment in the lab includes thirty-four Applied Biosystems 9700 GeneAmp PCR Systems for DNA amplification; BioRad iCycler Thermal Cycler for real-time quantitative PCR; a BioRad Gel Doc 1000 Video Gel Imaging System for gel documentation; a Sorvall Ultra-80 ultracentrifuge for DNA, RNA and protein preparation; a Jouan KR422 refrigerated microplate centrifuge for 96-well sample processing, as well as heating/cooling blocks; a refrigerated microcentrifuge; gel electrophoresis power supplies and chambers; and digital programmable multichannel repeat pipettors. A 96-well format Thermo Labsystems Fluoroskan Ascent fluorometer and the AutoGenprep 2000 System DNA extraction robot are used for extracting and quantifying large numbers of DNA/RNA samples. Several high-throughput robotic sample handling systems, the Beckman-Coulter Biomek 2000 Automated Workstation and Robbins Scientific Hydra 96 for liquid handling; and an Applied Biosystems 3730XL DNA Analyzer for capillary electrophoresis sequencing, SNP detection, and genetic fragment analysis facilitate our ability to process large numbers of samples. Our microarray facility is housed in a new ‘clean room’ and includes a robotic microarray pin spotter from Cartesian Technologies (model PixSys 5500); a Microgrid II robotic arrayer (Apogent Discoveries); the ScanArray® 5000 Microarray Analysis System (GSI Lumonics), equipped with four lasers capable of detecting most commercially available fluorescent dyes; an Array WoRx CCD-Scanner (Applied Precision).
Bioinformatics and computing facilities include a Sun Blade 2000 workstation with dual 900-MHz UltraSPARC III Cu Processors, a SunPCi IIpro CoProcessor, 4-GB RAM, and two 73-GB hard drives; 1 Ultra30 SUN workstation and 1 Sunblade 1000 Sun workstation, and a newly acquired 56 processor supercomputer running HP True 64 UNIX. Installed packages include GCG, EMBOSS, PHRED/PHRAP/CONSED, Sequencher, Gelcompare, ImaGene, QuantArray, MEGA, and BLAST.
The Greenhouse and Growth Chamber facilities include two new greenhouses that are in the final stages of construction. Each has a nominal floor space of 1500 sq. ft. and independent environmental control. The control system includes heating, cooling, shade curtain, growth lights, and roof vent that can be operated locally or remotely. Twenty-four high intensity 1000w growth lights are arranged in an overhead array of intermixed high pressure sodium and metal halide lamps to broadly span PAR and visible light spectrums. Each row (6) of 4 lights can be independently controlled. Temperature control is achieved using a chilled water/mechanical cooling system and steam heat designed for temperature control ± 3°C. Control system includes an outdoor weather station complete with sensor inputs for precipitation detection, solar radiation, air temperature, humidity, wind speed, and wind direction. A drip irrigation system (manually controlled currently) featuring an in-line fertigation system is included in each greenhouse. The system can currently accommodate up to 2,300 4" diameter pots. A 950 sq. ft. head-house provides adequate space for plant/soil preparations, and includes root washing station, sink/casework, and shelving. High-bay renovations include two plant growth chambers, walk-in coldroom, sample storage area (~10 ft x 10 ft), new metal halide, high intensity lighting, and an additional ~ 35 ft bench space/cabinetry. Ovens for drying plant and soil samples are available. In addition to greenhouse facilities, two walk-in plant growth rooms are available for use in this project. These growth rooms (Convirons Model BDW80) have an interior area of approximately 80 square feet.
The Microbiology Laboratory has facilities for work with aerobic and anaerobic microorganisms. The laboratory contains sterile transfer hoods, autoclaves, spectrophotometers, gas chromatographs, automated plating equipment, automated bacterial identification systems, laser colony counting equipment, fume hoods for work with volatile contaminants, gassing stations for the preparation of anaerobic media, two anaerobic glovebags (Coy Laboratory, Inc.), electrolytic respirometers, flow cytometry, an automated GeneTAC™ colony picker (Genomic Solutions), a robotic liquid handler (RoboSeq® 4204 SE), HiGro System (GeneMachines), and 3 Bioflo 110 modular benchtop chemostats (New Brunswick). Trained laboratory technicians keep this equipment operational.
Brookhaven National Laboratory
The Plant Physiology and Biochemistry lab has all the equipment needed to conduct physiological and biochemical analysis detailed in this proposal, including the cryogenic shipping and storage of plant material from ORNL. In addition to temperature controlled centrifuges, incubaters, sample concentrators, heating blocks and other standard laboratory equipment Dr. Rogers’ laboratory is equipped with a high performance liquid chromatography (HPLC) system consisting of a vacuum degasser, binary pump, thermostatted autosampler and column compartment, and a fluorescence detector (Agilent Technologies, 1100). This HPLC system is used exclusively for amino acid analysis. A plate reader capable of reading absorbance, luminescence and fluorescence (Biotek, Synergy HT) is used for analysis of chlorophyll, carbohydrates, protein, nitrate, nitrite, total amino acid content and enzyme activity assays. A CHNS/O elemental analyzer configured for CHN analysis (Perkin Elmer, Series II 2400) and a high precision analytical balance (Metler Toledo AX26DR) is used for determining elemental carbon and nitrogen content. Three portable infrared gas analyzers (2 x Li-6400, LiCOR; CIRAS-1, PP-Systems) and a Walz PAM2000 portable fluorimeter will be available for periodic physiological measurements at ORNL.
Los Alamos National Laboratory
The Microbial Ecology Facilities at LANL contains all of the equipment and infrastructure necessary for the proposed experiments. This includes five labs in the Health Research Laboratory (2,250 sq ft). These are equipped with six MJ Research thermal cycler units for PCR, two BioRad iCyclers for real-time PCR, two chemical fume hoods, a variety of bacterial shaker incubators, microwaves and standard ovens, heat blocks, water baths, vortexers, refrigerators (3), -20 freezers (4), -70C freezers (2), refrigerated floor and table-top centrifuges to accommodate microcentrifuge tubes as well as microtiter plates and test tubes/bottles up to 300 ml (6 total), electroporator, variable wavelength spectrophotometer and fluorometer, equipment for DNA extraction including dry baths, bead mill homogenizers (3), 96-well plate Genogrinder, microtiter plate spectrophotometer, microscope, pH meter, and balances. We also have a variety of electrophoresis units for mini and large scale agarose and acrylamide electrophoresis (one and two dimensional PAGE, DNA sequencing gels, DNA agarose gels, pulse field gels), and gel electroelution. We have three BSL2 biological safety cabinets, one ABI 3100 (16 port) sequencer, two ABI 377 sequencers, a Stratagene EagleEye gel imaging station, a Speed Vacuum system, and an Olympus Vanox-T epifluorescence microscope. Ultracentrifuges and liquid nitrogen dispensers are available nearby. Computing facilities include a Sun Sparc Ultra10 workstation, ten Macintosh computers, and two PC computers with network capability. In addition, each of the sequencing instruments and the iCycler is equipped with either a Mac or IBM workstation.