Phenotype MicroArrays for Microbial Cells
Supplied By: Biolog, USA
Phenotype MicroArrays for Microbial Cells Systems & Consumables
Phenotype MicroArray™ Technology enables researchers to characterize cells in up to 1,920 assays and evaluate cell changes under thousands of culture conditions and physiological states in a simple, rapid, efficient and cost-effective manner. By measuring a cell’s response to a genetic or environmental alteration, this integrated system of cellular assays, instrumentation, and bioinformatics software reveals invaluable information to speed insight and discovery and expedite scientific publication. Phenotype MicroArrays (PMs) have broad applicability for genotype-phenotype characterization as well as for determining optimal conditions for cellular growth, sporulation and germination, production of secondary metabolites, or enzymatic activities in cell lines.
The Biolog OmniLog® incubates and monitors 50 microplates, or 1,920 phenotypic assays simultaneously to measure physiological responses in diverse microbial cells.
A Breakthrough in Technology
Phenotype MicroArrays (PMs) represent the third major technology, alongside DNA Microarrays and Proteomic Technologies, that is needed in the genomic era of research and drug development. Just as DNA Microarrays and Proteomic Technologies have made it possible to assay the level of thousands of genes or proteins all at once, Phenotype MicroArrays make it possible to quantitatively measure thousands of cellular phenotypes all at once.
Phenotype MicroArray technology enables researchers to evaluate nearly 2000 phenotypes of a microbial cell in a single experiment. Through comprehensive and precise quantitation of phenotypes, researchers are able to obtain an unbiased perspective of the effect on cells of genetic differences, environmental change, and exposure to drugs and chemicals. You can:
Correlate genotypes with phenotypes
Determine a cell's metabolic and chemical sensitivity properties
Discover new targets for antimicrobial compounds
Optimize cell lines and culture conditions in bioprocess development
Characterize cell phenotypes for taxonomic or epidemiological studies
Important Applications of PM technology fall into three broad categories:
1. Testing Cell Lines Exposed to Drugs or Other Chemicals
Pharmaceutical companies and biotech companies spend much time, effort, and money screening and assessing drug candidates. This process could be made much more efficient and less costly with tools that can quickly and accurately assess issues such as (1) Sites and modes of action of drugs, (2) Drugs with high specificity versus drugs with side effects, and (3) Beneficial as well as detrimental interactions with other drugs. PM technology provides an ideal tool and can be used in all these ways to evaluate potential new drugs.
The experimental approach is very analogous to testing cells with genetic changes. In the case of genetic changes, a gene can be "knocked out" which usually means that a protein is not made (i.e., also "knocked out") and consequently some cellular function is blocked. Most drugs are targeted to "knock out" the function of a specific protein, so adding a drug to a cell should result in phenotypic effects that are very similar to "knocking" out the gene.
Drugs can be added to cells prior to inoculation into PMs. By looking at the phenotypes altered by the drug one can determine the physiological functions in the cell that are affected. This information will indicate: (1) the site and/or mode of action of a drug, (2) whether the drug is specifically hitting one target or whether it is interfering also with other cellular processes and therefore likely to cause side-effects, (3) potentially beneficial as well as detrimental interactions with other drugs (many of the phenotypes in the PMs test for increased sensitivity or resistance to existing drugs).
Toxicological testing based on the use of cell lines is gradually replacing animal testing as a more cost-effective and humane approach. Using PMs, testing would be performed essentially as described in the preceding paragraph for drug testing. One simply adds a chemical to the cells and inoculates into PMs. Interference with an aspect of cell physiology will be manifested as an altered response in the PMs. The information from PMs will indicate toxicity levels as well as mechanisms of toxicity. Some chemical agents may be toxic only under specific growth conditions or only in combination with other toxic chemicals. Since PMs contain thousands of different testing conditions, they provide toxicological information that is much more thorough and comprehensive.
2. Testing Cell Lines with Genetic Differences
Cells have on the order of 2,000 to 40,000 genes. Even in the simplest and most studied microbial cells, only about half of the genes have a known function. Through a variety of genetic and biochemical techniques, scientists are identifying many genes as being "especially interesting". For example, from studies of hereditary human genetics, these genes may be implicated in an important disease or syndrome. Alternatively, they may be involved in cancer or microbially-induced or chronic diseases and thereby be potential targets for new drugs. Genes of great importance (both biologically and commercially) are also being identified in other animals, plants, and microorganisms. However, with current technology it is very difficult, expensive, and not-at-all straightforward to determine the function of these important genes.
The method most commonly proposed for determining the function of unknown genes relies on the use of nucleic acid- based microarrays. DNA microarrays are used to measure mRNA levels under several growth conditions, and then the data are analyzed to see if the mRNA levels of the unknown gene go up and down in correlation with the mRNA levels of a known gene. The hope is that by grouping genes that are regulated in the same manner, biologists will be able to discern genes that are members of the same functional pathways. This approach is rather lengthy, expensive, and complex and it relies on making numerous assumptions that may be incorrect.
PMs make it possible to go directly from a gene of interest to a cellular function. The experimental approach is to simply "knock out" the gene of interest in a cell line to create an isogenic pair of strains. The biologist simply inoculates the isogenic cell lines into the PMs and looks for one or more phenotypic differences. Alternatively one can do a "knock in" genetic construction in which a gene of interest is added to a cell line. Here again, the isogenic cell lines are assayed in PMs and one looks for discernable phenotypic differences. By analyzing isogenic strains and using PMs in large scale, high-throughput studies we can start from a genomic map and generate a virtual phenotypic map. PM technology is fast, inexpensive, and simple, and it does not make assumptions about gene transcription, translation, or post-translational modifications.
Finding Genes that Code for New Drug Targets
PM technology can be used in comparisons of cell lines to find new drug targets. For example, in antimicrobial R&D, pairs of pathogenic and non-pathogenic microbial strains can be compared. Pathogenic strains are known to contain additional genes such as drug resistance genes and pathogenicity islands. These extra genes code for proteins that convey additional phenotypes to the microbe and may be useful as drug targets.
Another example is the search for new anti-cancer agents. Cancerous cells can be compared to non-cancerous cells in PMs to look for phenotypic sites of potential vulnerability.
A major objective of many animal and plant genetic projects is the improvement of targeted cell lines or seed lines. After genetic manipulation, cell lines must be evaluated to see if they picked up the desired phenotypic traits and also to see if they picked up any undesired secondary phenotypic traits. PM technology will clearly be a very useful tool in these types of developments.
Cell lines can and do change when they are subcultured. This is due, at least in part, to unstable genetic constructions and to selective pressures that biologists unknowingly apply to cultured cells. Cell line stability is an important issue in basic research (e.g. cancer research) and in important medical applications such as vaccine and recombinant protein production. It is essential to know when and in what ways cell lines are changing
3. Direct Testing of Cell Lines
There is a great deal of interest in basic research and applied development in optimizing conditions for growing cells, especially animal and plant cells. In basic research it is important to understand the growth requirements of cells so that they can be handled properly and cultured rapidly. In commercial ventures, cells are cultured in vitro for many applications including cell transplantation and gene therapy, tissue replacement, vaccine production, and recombinant protein production. Many of the PMs contain biochemicals that may act as nutrients for certain cell types. A stimulatory effect on the cells will be detected as an increase in respiration and therefore an increase in color from the redox dye. This will point the scientist toward nutrients that improve the growth and health of the cells.
Many plants and microorganisms can go through stages in their life cycle where they form seeds or spores and later germinate. The conditions that trigger these changes are often very difficult to discern, requiring a very precise combination of culture conditions. Knowing optimal conditions for sporulation and germination can have important economic benefits. Sporulation is important in basic research and genetic and physiological studies, for example with actinomycetes, yeasts, and filamentous fungi. Germination is important in agriculture for cultivation of mushrooms and plants. PM's will offer biologists a very simple and straightforward means to streamline this testing.
Secondary metabolites such as antibiotics and pigments typically are produced under very specific growth conditions, often involving some special limitation of growth. PMs can provide thousands of growth conditions and allow a very rapid and easy way to look for optimal production conditions.
Many of the phenotypes that are measured with PMs indicate the presence of enzymatic activities that may have potential commercial use. Examples are enzymes involved in catabolism of carbon, nitrogen, phosphorus, and sulfur, and enzymes that protect cells against toxic chemicals. Therefore PMs can be useful for screening microorganism collections to look for these activities.
PMs provide an easy and highly efficient means for testing cells under thousands of diverse growth conditions. It provides a flexible and versatile format for many other types of cell-based experiments and assays. The limits are defined by the creativity and imagination of the scientist using them.
Phenotype MicroArrays for Microbial Cells
Phenotype MicroArray Technology is now available for applications with nearly all important species of bacteria and fungi. Simple protocols are available for testing nearly 2000 cellular phenotypes simultaneously.
Uses with microbial cells
Determine gene functions
Compare gene knock-out mutants to wild types
Compare naturally-isolated strains with different genetic backgrounds
Identify novel antimicrobial targets by finding genes unique to pathogenic Micro Organisms
Find phenotypes present in pathogenic but not in non-pathogenic strains
Find phenotypes present in pathogenic microbes but not in host cells (animal or plant)
Test antimicrobial targets and drug leads by comparative phenotyping
Determine MOA of drug leads
Compare phenotypic changes caused by target gene knockout versus drug addition
Compare and analyze different generations of production strains
QC fingerprint production strains
Scan 2000 culture conditions simultaneously to optimize the growth medium for product yield
General cell characterization
Determine metabolic properties of any microbe
Determine drug/chemical sensitivities
Available tests for microbial cells
To view the tests in the PM sets, click on the map links below.
PM-1 to PM-10: Metabolic tests for bacteria and fungi
PM-11 to PM-20: Chemical sensitivity tests for bacteria
PM-21 to PM-25: Chemical sensitivity tests for fungi
Drug Discovery Using PMs
The Drug Discovery Process
PMs can be applied at multiple stages in the drug discovery process, providing researchers with better information to help make better decisions. With PMs the entire process becomes faster and more efficient.
The current drug discovery and development process is long and expensive. Just consider:
Only one compound of 10,000 makes it to market
It can take upwards of 15 years and $800 million to commercialize a single drug
The challenge for drug companies is three-fold: reduce product development costs… decrease time to market… and increase the probability of success for the most promising leads. Biolog's PM technology can assist with these three challenges — and more.
The genomic revolution has delivered massive amounts of data about life's molecular components, moving the bottleneck in drug discovery downstream by giving drug discovery organizations more qualified targets and leads than ever before. However, there remains a barrier in going from molecular data to understanding what really happens in living cells. PMs provide the needed technology to complement molecular genomics and cross that barrier (see diagram below).
Just as genomics and proteomics have made it possible to measure levels of sounds of genes and proteins all at once, Biolog's new PM technology can quantitatively measure thousands of cellular phenotypes in one analysis. This provides the technological transition over the barrier from molecular to cellular analysis.
Impact on the Drug Discovery Process
Continuing innovations in genomics and analytical technologies have made it particularly difficult for pharmaceutical and biotechnology organizations to determine the optimum approach to employ in the drug discovery process. Biolog has demonstrated that Phenotype MicroArray technology has the ability to deliver significant benefits at multiple stages in the drug discovery process:
Permits direct cellular assay of gene function
Complements genetic and molecular methods and extends existing gene expression data
Provides analysis of genetic mutations
Identifies metabolic pathways and may include enzyme specific targeting
Allows efficient evaluation of drug leads
Allows direct observation of a drug's primary and secondary effects at the cellular level
Reduces need for expensive, slow animal / plant models
The result of using PM technology? More promising druggable leads and a reduction in failure rates. PMs help drug companies to choose the right drug leads for animal and human testing, saving time and money by lowering the opportunity cost associated with bad decisions.
Brochure on Using PM Technology in Drug Discovery
Click Here to view the document providing an overview of PM technology applied to drug discovery
Mechanism of Action (MOA) of Antimicrobials
Click Here to view the document on Using PM Technology in Antimicrobial MOA Studies
Structure Activity Relationship (SAR) Analysis
Click Here to view a document on Using PM Technology to Guide SAR Studies
How PM Technology Works
Phenotype MicroArrays (PMs) represent the third major technology, alongside DNA Microarrays and Proteomic Technologies, that is needed in the genomic era of research and drug development (Figure 1). Just as DNA Microarrays and Proteomic Technologies have made it possible to assay the level of thousands of genes or proteins all at once, Phenotype MicroArrays make it possible to quantitatively measure thousands of cellular phenotypes all at once. Many publications demonstrate the power of this technology in enabling new discoveries and in generating new hypothesis.
DNA Microarrays and Proteomic Technologies allow scientists to detect genes or proteins that are coregulated and whose patterns of change correlate with something important such as a disease state. However there is no assurance that these changes are really significant to the cell. Phenotype MicroArrays are a complementary technology providing the needed information at the cellular level ... and much more.
Phenotype MicroArrays provide comprehensive cellular profiles that can be used to identify gene function, validate drug targets, and streamline lead validation, optimization, and toxicology studies. After a genetic change or exposure to a drug lead, the researcher can directly evaluate the cellular response to that change.
Phenotype MicroArray technology is a breakthrough platform technology. It is an integrated system of cellular assays, instrumentation, and bioinformatic software for high-throughput screening (HTS) of cells.The technology and the testing process are shown Figure 2. Biolog preconfigures a wide range of phenotypic tests into sets of arrays. Each well of the array is designed to test a different phenotype. The scientist simply inoculates a standardized cell suspension into the wells of the MicroArray, thereby testing thousands of phenotypes at once. The MicroArray is then incubated, typically for 24 hours.
PMs use Biolog's patented redox chemistry, employing cell respiration as a universal reporter. If the phenotype is strongly "positive" in a well, the cells respire actively, reducing a tetrazolium dye and forming a strong color (Figure 2, left). If it is weakly positive or negative, respiration is slowed or stopped, and less color or no color is formed. The redox assay provides for both amplification and precise quantitation of phenotypes.Incubation and recording of phenotypic data is performed by the patented OmniLog instrument (Figure 2, middle) which captures a digital image of the MicroArray several times each hour and stores the quantitative color change values into computer files. The computer files can be displayed to the scientist in the form of kinetic graphs. Thousands of phenotypes are monitored simultaneously by the OmniLog and up to 450,000 data points can be generated in one 24-hour run. To compare the phenotypes of two cell lines, one is recorded as a red tracing and one as a green tracing (Figure 2, right). These graphs can then be overlaid by the bioinformatic software to detect differences. Areas of overlap (i.e. no change) are colored yellow, whereas differences are highlighted as patches of red or green (Figure 2, right and Figure 3).
Phenotype MicroArrays can monitor, either directly or indirectly, most known aspects of cell function. The range of phenotypes includes:
Cell surface structure and transport functions
Catabolism of carbon, nitrogen, phosphorus, and sulfur
Biosynthesis of small molecules
Synthesis and function of macromolecules and cellular machinery
Cellular respiratory functions
Stress and repair functions
Other cellular properties