Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-01
2002-06-25
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S243000, C435S252300, C435S254110, C435S320100, C435S007100, C435S030000, C435S252100, C435S261000
Reexamination Certificate
active
06410244
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method for preparing plasmids, particularly a method amenable for use with high-throughput library screening.
BACKGROUND OF THE INVENTION
Current processes for producing plasmids for screening generally involve plating a large number of colonies, culturing the colonies, and scraping the colonies into microtubes for analysis. This process, however, can be labor intensive, because a researcher must manually transfer colonies to the microtubes. Furthermore, the scraping process may break cells, resulting in a viscous solution, and also creates a substantial risk of contamination from adventitious microorganisms. Thus, this process limits the rate and reliability of gene expression library screening. A convenient and simple method for preparing a plasmid pool suitable for high-throughput screening would substantially increase productivity and efficiency of the screening process.
SUMMARY OF THE INVENTION
The present invention generally relates to a method for culturing a plasmid library by combining a plasmid library comprising a plurality of microorganisms, each transfected with one of a plurality of plasmids or other nucleic acid constructs, with a semi-solid or gelled culture medium, dividing the culture medium between at least ten receptacles (e.g., wells), incubating the receptacles, and separating the microorganisms from the culture medium. In certain embodiments, the method includes isolating DNA from the microorganisms.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of preparing plasmids wherein a plasmid library, e.g., a variegated library of genetic constructs (plasmids, vectors, etc.), is mixed with a culture medium which is then divided among a plurality of reservoirs. The culture medium is preferably a semi-solid matrix, e.g., a polymer gel, such as agar, agarose, gelatin, methylcellulose, etc. After culturing, the cells may be separated from the culture medium, e.g., by centrifugation or filtration, and the plasmids may be prepared by techniques known in the art. Because the individual cultures are separate from each other, preferably in a predefined arrangement, the final preparation process may be automated, e.g., by using a robot.
I. Definitions
Before further description of the preferred embodiments of the subject invention, certain terms employed in the specification, examples, and appended claims are collected here for convenience. “Culture medium” refers to a liquid, semi-solid, or solid solution including sufficient nutrients for supporting the growth of microorganisms.
The terms “gel” and “semi-solid” are used herein to refer to a material, such as a polymer or other crosslinked (e.g., covalently or non-covalently) matrix, in which the diffusion of particles, such as microorganisms, is substantially impeded relative to in a liquid.
“Gelling agent” refers to a compound which can be added to a liquid to thicken liquid, for example, to provide a solid or semi-solid material, such as a gel. Common gelling agents include agar and agarose.
As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. ESTs, chromosomes, cDNAs, mRNAs, and rRNAs are representative examples of molecules that may be referred to as nucleic acids.
The term “plasmids” refers generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, “plasmid” and “vector” are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
A “plasmid library” or “expression library,” as the terms are used herein, refers to variegated library of transformed cells, e.g., a plurality of microorganisms each expressing a plasmid, wherein a plurality of different plasmids, e.g., at least 10, preferably at least 100, even more preferably at least 1000, 10,000, or more, are represented in the plurality of microorganisms.
“Selection agent” refers to a compound added to a culture medium which inhibits the growth of microorganisms which lack a particular gene. For example, ampicillin is a selection agent which inhibits the growth of microorganisms which lack an ampicillin resistance gene.
“Recovery” or “isolation” of a given DNA means separation of the DNA from a host cell.
The terms “replicable expression vector” and “expression vector” refer to a piece of nucleic acid, e.g., DNA (usually double-stranded), which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of the host chromosomal DNA, and several copies of the vector and its insert (foreign) DNA may be generated. In addition, the vector may, if appropriate, contain the necessary elements that permit translating the foreign DNA into a polypeptide.
The terms “transformed host cell” and “transformed” refer to the introduction of DNA or other nucleic acids into a cell. The cell is termed a “host cell”, e.g., a prokaryotic cell. Typical prokaryotic host cells include various strains of
E. coli.
The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the same species as the host cell or a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign and some homologous DNA.
II. Methods of the Present Invention
The present invention generally relates to methods for preparing libraries of nucleic acid constructs by amplifications using transfected microorganisms. Generally, microorganisms of an expression library are added to culture medium to which a gelling agent and, optionally, a selection agent is added. Preferably, the concentration of microorganisms in the culture medium is between 1 and 1000 colony-forming units (CFU) per mL, more preferably between 10 and 500 cfu/mL, and even more preferably between 50 and 400 cfu/mL. The culture medium is then divided among a plurality of reservoirs, such as wells, test tubes, vials, or other suitable containers. The culture medium containing the microorganisms is then incubated and cultured according to techniques well known in the art. The resulting colonies of microorganisms may then be separated from the growth medium, e.g., by centrifugation, filtration, or other suitable means. The resulting cells may then be processed to prepare the plasmids, e.g., by lysing the cells, purifying the plasmid DNA, etc. The processing may be automated, e.g., by using a robot.
Dividing the growth medium among a plurality of reservoirs has several advantages. For example, using standard plating techniques, colonies of cells may be overgrown and/or crowded out by neighboring colonies. Elimination of colonies in this fashion skews the proportions of plasmids detected, potentially affecting the outcome of the experiment. Using separated reservoirs, however, overgrowth is substantially curtailed, preserving the representation of plasmids in the original population of microorganisms. The use of a semi-solid or gelled matrix further hampers overgrowth, because each colony is relatively isolated in three-dimensional space, restricting the possibility of overgrowth into a neighboring colony. Additionally, using standard plating techniques, the colonies must be removed from the plate by hand and transferred into receptacles for further pro
Curis, Inc.
Guzo David
Halstead David P.
Ropes & Gray
Vincent Matthew P.
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