Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-04-21
2001-06-19
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007210, C435S325000, C435S243000, C435S252300, C435S376000, C435S029000, C435S030000, C435S034000, C435S375000, C435S007400
Reexamination Certificate
active
06248541
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to methods for obtaining populations of cells in culture having similar density. It is also directed to methods of screening populations of cells for a phenotype of interest, including improved production of products and production of improved products.
BACKGROUND OF THE INVENTION
Microorganisms are being widely used as sources of useful chemical products, whether naturally occurring or through genetic engineering. Screening of organisms, particularly single cell organisms, is necessary for identifying those that produce useful products or useful products in more abundant quantities. Such identified organisms may then be isolated and utilized for their useful attributes.
Traditionally, screening of a population of organisms has been performed on agar plates. The population is usually spread on plates containing an indicator for a target enzyme, for example a chromogenic substrate, or skim milk to detect proteases. Colonies of the plated organism which produce elevated levels of the target enzyme can be detected, based on the intensity of color, the clearing zone, etc.
The method of screening by plating on agar plates has several disadvantages. The conditions on agar plates are very different from production conditions, which generally involve high-density fermentation. Therefore, a strain of an organism which produces elevated levels of a product on agar will not necessarily do so during production. Furthermore, the intensity of a reporting signal on an agar plate is dependent on the size of a given colony, masking variations in productivity between strains. Agar plate screens usually favor fast-growing strains which tend to give stronger signals.
The analysis of microcolonies has recently been described (Yang et al.,
Gene
173:19-23 (1996)). This method allows screening of large numbers of colonies on a single agar plate. However, this method still suffers from the problems mentioned above.
More recently, screening of populations of organisms has been performed on microtiter plates. This method generally involves distributing individual clones into wells of microtiter plates, for example by using a colony picker. The cells are then grown in a rich medium which mimics production conditions, allowing cells to grow to a high density. The cells are allowed to grow for various lengths of time. Samples are withdrawn for analysis, frequently requiring dilution of the sample to bring the activity of the target enzyme into the range of the detection method. Activity can be measured by various means, e.g., using chromogenic or fluorogenic substrates, radioactivity, etc.
The microtiter screening methods described above have several disadvantages. They require several liquid handling steps, which limits the throughput and/or requires robotics, which is very expensive. Liquid handling steps are also sources of error. It is also difficult to provide cultures in microtiter plates with oxygen, which is essential for many hosts. Furthermore, the cultures grow to a high density, resulting in changing medium conditions, such as pH, nutrient concentrations, by-products, etc. The changing medium conditions affect the productivity of the cells, resulting in the identification of strains which produce well under such conditions, but not necessarily under production fermentation conditions.
Therefore, there is a need for a rapid and efficient and cost-effective means for screening large numbers of cells for individual strains having preferred productivity of a product under conditions that closely approximate production fermentation conditions. Such a screen would be useful for identifying not only cells of a population with naturally occurring preferred properties, but also for identifying such preferred members of a population resulting from induced mutagenesis.
SUMMARY OF THE INVENTION
The present invention provides novel methods for obtaining a plurality of populations of cells having similar density and screening cell populations for a phenotype of interest, including improved production of products and production of improved products. In one aspect, the method comprises culturing a plurality of samples from an initial population of cells, in a medium containing at least one nutrient in limiting concentration. Growth of cells in each sample is controlled by the limiting nutrient to produce subpopulations having a similar cell density. This greatly simplifies the comparison of the subpopulations. In one embodiment, the cells are from a single initial population which may be a single organism type or cell line or a pool of preferred cells. The samples may be inoculated at various cell densities by various methods. Different limiting nutrients may be used, and the samples may be cultured in various receptacles.
In addition, the present invention provides methods for screening a population of cells of interest for cells having a phenotype of interest. The method comprises obtaining from the cell population of interest a plurality of subpopulations of cells of similar density by culturing samples of the cell population of interest. The samples comprise cells from the population of interest and a cell culture medium containing a limiting nutrient that controls cell growth such that cells in each sample grow to a similar density. Subpopulations comprising cells having the phenotype of interest are then identified.
In another embodiment, the invention provides methods of screening a population of cells of interest for cells having the phenotype of improved production of one or more products of interest. This method comprises the steps of obtaining from the cell population of interest a plurality of subpopulations of cells of similar density by culturing samples of the cell population of interest. The samples comprise cells from the population of interest and a cell culture medium containing a limiting nutrient that controls cell growth such that cells in each sample grow to a similar density. The amount of the product produced by the individual subpopulations is then measured. In a preferred embodiment, the improved production is during stationary phase. In a preferred embodiment, improved production is increased total production or increased production over time. Preferably, the amount of the product is measured. In one embodiment, measuring is by measuring activity of the product in each of the plurality of cell populations. In a preferred embodiment, the samples contain or have added to them a substance that detects the presence of each of the products. In one preferred embodiment, the substance is a substrate of the product. In another preferred embodiment, the substance is a ligand of the product. Preferably, the substance is fluorogenic or chromogenic.
Also provided by the invention is a method of screening a cell population of interest for cells having the phenotype of producing an improved product of interest. In one aspect, the improved product has increased activity over an unimproved product. The method comprises obtaining a plurality of subpopulations from the population of interest by culturing samples from the population of interest with at least one limiting nutrient which controls cell growth. In a preferred embodiment, the activity of the product is measured in the subpopulations to determine the relative activity of the product made by each.
In addition, the invention provides a method of screening a cell population of interest for cells having the phenotype of producing a product of interest having altered selectivity of substrate(s). The method comprises obtaining a plurality of subpopulations from the population of interest by culturing samples from the population of interest with at least one limiting nutrient which controls cell growth. The samples contain or have added to them, together or sequentially, two or more different substrates for the product of interest. In a preferred embodiment, the activity of the product on each substrate is measured in the subpopulations. Preferably the substrates are fluorogenic, are distinguish
Genencor International Inc.
Guzo David
Stone Christopher L.
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