Detailed Research Information

Detailed project information for
Study Plan Number 01103

Branch : Fish Health Branch

Study Plan Number : 01103

Study Title : Development of Fluidized Microarray Technology for the Study of Function and Ecology of Environmental Microbes

Starting Date : 08/01/2003

Completion Date : 09/30/2006

Principal Investigator(s) : Schill, Bane (William)

Primary PI : Schill, Bane (William)

Telephone Number : (304) 724-4438

Email Address : bane_schill@usgs.gov

SIS Number :

Primary Program Element : Fisheries and Aquatic Resources

Second Program Element :

Status : Completed

Abstract : BACKGROUND
Bacteria are ubiquitous inhabitants of all sorts of environments including fresh and salt waters, thermal vents, arctic snow, acidic mine drainage, petroleum deposits, soils, and sediments. Recent studies demonstrate that bacteria communicate with each other through chemical messaging and even communicate with the cells of plant and animal hosts to modulate physiology, development, and the immune system. Large shifts in microbial populations can be triggered by subtle environmental cues, but once begun, these shifts may themselves cause dramatic ecosystem changes. This is because microbes are responsible for many geochemical processes including metal and sulfur oxidation and reduction, nitrification and denitrification, fermentation, and methane production. Over 1500 scientific studies (PubMed search) have been published using small subunit ribosomal RNA to elucidate shifts in microbial assemblages in response to particular environmental stressors. The great majority of those have utilized genetic sequencing of ribosomal RNA to identify the bacteria present and their distribution. While the advent of high throughput automatic sequencing has made these studies feasible, bacteria react quickly to environmental insults, and the application of the sequencing approach to repetitive samples rapidly becomes impractical. What is needed is a method that will allow for the measurement of the presence and/or activity of thousands of genes simultaneously. Such a platform, known as the microarray, is currently available but has not been widely applied to the study of environmental bacterial assemblages. Although microarrays are usually formatted as a grid of hybridization probes spotted onto microscope slides, this is not without inherent problems. Production of the microarray slide requires robotics to apply the probes precisely, and applied probes (“features”) may be subject to artifacts such as non-uniformities in spot morphology and surface blemishes. Additionally, one slide is one test. For statistical purposes, repetitions must be performed. Several of these weaknesses can be alleviated by changing the format of the array from two-dimensional to a three-dimensional fluidized bead format. Sets of microscopic (5m) latex beads are doped with mixtures of fluorescent dyes in varying amounts to created a substrate (fluorescently “bar-coded” bead set) that can be identified by its characteristic fluorescent signature. A hybridization probe is then covalently coupled to the surface of the bar-coded bead creating the equivalent of one feature (spot) on a standard two-dimensional microarray slide. Numerous bead-probe combinations can be produced in this way to create a fluidized microarry with each bead and its associated probe identified by the fluorescent signature of the bead. Simultaneous quantification of each of a number of targets (DNA or RNA species homologous to the probes on the beads) is possible when target mixtures labeled with yet another fluorescent reporter molecule are hybridized to mixtures of probe-bead sets. Beads bearing probes with hybridized targets are decoded and hybridized targets are quantified by the familiar technique of flow cytometry. Fluorochromes are excited as each bead passes through a laser beam. Photomultiplier detectors with filters tuned to the emission wavelengths of the fluors used to dope the beads detect the passage of a bead and its bar-code while another tuned photomultiplier detector measures the amount of target hybridized to the bead in the laser beam. Numerous beads of different types with their associated hybridized targets are detected, decoded, and quantified in this way. Thus, each bead of a particular type that is scored equates to a repetition of the quantification of the associated analyte. This allows good statistical confidence in the results. Also, problems of feature artifacts typical in two-dimensional arrays are eliminated with fluidized bead arrays because of the surface uniformity of the bead as well as because any nonhomogeneity is minimized due to its spin as it passes through the laser path.
OBJECTIVES
I will develop a robust suite of probes suitable for use in fluidized microarray (www.luminexcorp.com) methodology to be used to identify and quantify the taxonomic groups of bacteria present in environmental samples in near real-time. This goal will be approached by (1) using newly developed, innovative computer programs to analyze the public sequence databases and produce signature sequences unique to specific taxonomic groups, (2) design optimal synthetic oligonucleotide probes using thermodynamic modeling, and (3) verify the performance of the taxonomic probes for fluidized microarray use by testing performance of probe sets attached to microbeads coded by fluorescent labels and interrogated by laser scanning cytometry (www.compucyte.com). Microarrays will be tested for utility in monitoring shifts in microbial populations in freshwater (surface and groundwater) and marine (turtle grass bed and coral) environmental situations.
HYPOTHESES TO BE TESTED
1. Hybridization probes can be identified by computer analysis of database sequences that specifically target defined taxonomic groups of bacteria.

2. Probes identified in this way perform as predicted in empirical tests.

3. Suites of suitable probes can be combined into microarrays of fluidized format for following shifts in bacterial consortium species composition resulting from environmental insults.

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11700 Leetown Road, Kearneysville, WV 25430, USA
URL: http://www.lsc.usgs.gov
Maintainer: lsc_webmaster@usgs.gov
Last Modified: October 21, 2002 dwn
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Branch :	Fish Health Branch
Study Plan Number :	01103
Study Title :	Development of Fluidized Microarray Technology for the Study of Function and Ecology of Environmental Microbes
Starting Date :	08/01/2003
Completion Date :	09/30/2006
Principal Investigator(s) :	Schill, Bane (William)
Primary PI :	Schill, Bane (William)
Telephone Number :	(304) 724-4438
Email Address :	bane_schill@usgs.gov
SIS Number :
Primary Program Element :	Fisheries and Aquatic Resources
Second Program Element :
Status :	Completed
Abstract :	BACKGROUND Bacteria are ubiquitous inhabitants of all sorts of environments including fresh and salt waters, thermal vents, arctic snow, acidic mine drainage, petroleum deposits, soils, and sediments. Recent studies demonstrate that bacteria communicate with each other through chemical messaging and even communicate with the cells of plant and animal hosts to modulate physiology, development, and the immune system. Large shifts in microbial populations can be triggered by subtle environmental cues, but once begun, these shifts may themselves cause dramatic ecosystem changes. This is because microbes are responsible for many geochemical processes including metal and sulfur oxidation and reduction, nitrification and denitrification, fermentation, and methane production. Over 1500 scientific studies (PubMed search) have been published using small subunit ribosomal RNA to elucidate shifts in microbial assemblages in response to particular environmental stressors. The great majority of those have utilized genetic sequencing of ribosomal RNA to identify the bacteria present and their distribution. While the advent of high throughput automatic sequencing has made these studies feasible, bacteria react quickly to environmental insults, and the application of the sequencing approach to repetitive samples rapidly becomes impractical. What is needed is a method that will allow for the measurement of the presence and/or activity of thousands of genes simultaneously. Such a platform, known as the microarray, is currently available but has not been widely applied to the study of environmental bacterial assemblages. Although microarrays are usually formatted as a grid of hybridization probes spotted onto microscope slides, this is not without inherent problems. Production of the microarray slide requires robotics to apply the probes precisely, and applied probes (“features”) may be subject to artifacts such as non-uniformities in spot morphology and surface blemishes. Additionally, one slide is one test. For statistical purposes, repetitions must be performed. Several of these weaknesses can be alleviated by changing the format of the array from two-dimensional to a three-dimensional fluidized bead format. Sets of microscopic (5m) latex beads are doped with mixtures of fluorescent dyes in varying amounts to created a substrate (fluorescently “bar-coded” bead set) that can be identified by its characteristic fluorescent signature. A hybridization probe is then covalently coupled to the surface of the bar-coded bead creating the equivalent of one feature (spot) on a standard two-dimensional microarray slide. Numerous bead-probe combinations can be produced in this way to create a fluidized microarry with each bead and its associated probe identified by the fluorescent signature of the bead. Simultaneous quantification of each of a number of targets (DNA or RNA species homologous to the probes on the beads) is possible when target mixtures labeled with yet another fluorescent reporter molecule are hybridized to mixtures of probe-bead sets. Beads bearing probes with hybridized targets are decoded and hybridized targets are quantified by the familiar technique of flow cytometry. Fluorochromes are excited as each bead passes through a laser beam. Photomultiplier detectors with filters tuned to the emission wavelengths of the fluors used to dope the beads detect the passage of a bead and its bar-code while another tuned photomultiplier detector measures the amount of target hybridized to the bead in the laser beam. Numerous beads of different types with their associated hybridized targets are detected, decoded, and quantified in this way. Thus, each bead of a particular type that is scored equates to a repetition of the quantification of the associated analyte. This allows good statistical confidence in the results. Also, problems of feature artifacts typical in two-dimensional arrays are eliminated with fluidized bead arrays because of the surface uniformity of the bead as well as because any nonhomogeneity is minimized due to its spin as it passes through the laser path. OBJECTIVES I will develop a robust suite of probes suitable for use in fluidized microarray (www.luminexcorp.com) methodology to be used to identify and quantify the taxonomic groups of bacteria present in environmental samples in near real-time. This goal will be approached by (1) using newly developed, innovative computer programs to analyze the public sequence databases and produce signature sequences unique to specific taxonomic groups, (2) design optimal synthetic oligonucleotide probes using thermodynamic modeling, and (3) verify the performance of the taxonomic probes for fluidized microarray use by testing performance of probe sets attached to microbeads coded by fluorescent labels and interrogated by laser scanning cytometry (www.compucyte.com). Microarrays will be tested for utility in monitoring shifts in microbial populations in freshwater (surface and groundwater) and marine (turtle grass bed and coral) environmental situations. HYPOTHESES TO BE TESTED 1. Hybridization probes can be identified by computer analysis of database sequences that specifically target defined taxonomic groups of bacteria. 2. Probes identified in this way perform as predicted in empirical tests. 3. Suites of suitable probes can be combined into microarrays of fluidized format for following shifts in bacterial consortium species composition resulting from environmental insults.
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U.S. Department of the Interior \|\| U.S. Geological Survey 11700 Leetown Road, Kearneysville, WV 25430, USA URL: http://www.lsc.usgs.gov Maintainer: lsc_webmaster@usgs.gov Last Modified: October 21, 2002 dwn Privacy Policy and Disclaimers \|\| FOIA \|\| Accessibility