Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1999-07-14
2003-09-16
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S243000, C435S252100, C435S252300, C435S252330, C435S254100, C435S254200, C435S256800, C435S260000, C435S285100, C435S404000, C435S822000
Reexamination Certificate
active
06620609
ABSTRACT:
FIELD OF THE INVENTION
Ongoing advances in nucleic acid (DNA and RNA) molecular biology have greatly contributed to the growth of knowledge in all fields of biology. The entire body of traditional disciplines in biology has benefited from this molecular biology contribution. Mention may be made of microbiology and its branches: bacteriology, mycology, parasitology and virology, but also of cellular biology, cellular physiology, molecular genetics, biochemistry, enzymology, immunology and animal physiology. Use of the potentials of nucleic acids has resulted in the appearance of new, hitherto unknown fields of investigation known by new terms such as genomics, molecular medicine, and gene therapy.
Recombinant DNA technologies have grown just as rapidly thanks to the use of the bacterium
Escherichia coli
. The K12 strain of
Escherichia coli
together with its many mutants has, over the years, become an essential intermediary in all operations involving nucleic acids. Any researcher using recombinant DNA in almost all fields of biology is led to work with this bacterium, particularly in the form known as transformed, as a result of the introduction of plasmid or viral DNA into the cell. This is how, for example, the great majority of recombinant DNA preparations, which may include fragments of natural or modified DNAs of different origins obtained from the realm of procaryotic and eucaryotic, archeons or even chemically synthesized, are made from vectors specific to
Escherichia coli
. Laboratory strains of the yeast
Saccharomyces cerevisiae
must also be mentioned as it is one of the most frequently used microorganisms for the production of recombinant DNA.
The transformation of a bacterium or of a eucaryotic microorganism by a vector is an operation that is frequently performed by researchers and is performed in two stages. The first stage consists in introducing the vector's DNA into cells by means of various techniques, and the second stage in selecting those cells in the population that have received and express the vector's genes. The proportion of cells that express the vector's genes among the total number of cells varies greatly according to the type of transforming DNA and the transfer procedure used. Even in the best cases the population of transformed cells is always small. Several methods have therefore been devised to select only those cells that express the new gene or genes introduced by the transforming DNA. All rely on the expression of a gene whose product gives a dominant character to the cell relative to non transformed cells. An example is the use of a gene that is resistant to an antibiotic that is normally toxic to the receiving microorganism. The addition of a given antibiotic to a microbial cell that has been in contact with the DNA carrying the gene that is resistant to the antibiotic either prevents growth or kills cells that do not express the transgene, according to the nature of the antibiotic, whereas on the contrary the transformed cells that have become resistant multiply to finally produce a pure culture of resistant cells. For practical reasons selection is usually done with solid media in a Petri dish. In this case each transformed cell produces an independent pure culture that appears as the formation of colonies on the surface of the medium contained in the Petri dish. The most frequently used antibiotics for selecting transformed clones in
E. coli
are ampicillin and carbenicillin among the penicillins, kanamycin and neomycin among the aminosides, tetracyclin, chloramphenicol and zeocin.
The use of selection markers is often associated with the use of identification markers, in other words genes that enable transformed colonies to be distinguished from colonies that do not express said transgene by a different color. The most popular system among researchers is that based on the expression of the lacZ gene of the
Escherichia coli
&bgr;-galactosidase. The action of this enzyme, either whole or reconstituted as in the alpha peptide system used in many of the cloning vectors in
E. coli
leads to hydrolysis of a colorless compound, 5bromo-4-chloro-3-indoxyl-&bgr;-D-galactopyranoside (or X-gal) to give a blue insoluble product. The presence of this coloring agent in solid media for
E. coli
, for example, leads to the appearance of blue colonies formed by cells producing an active &bgr;-galactosidase, whereas cells devoid of &bgr;-galactosidase produce white colonies. Many other chromogenic or fluorescent substrates of &bgr;-galactosidase are used for the same purpose of visual distinction of positive populations among a population of white or non fluorescent colonies. Similar chromogenic or fluorescent substrates are also often used to detect positive colonies for other enzymatic activities such as &bgr;-glucuronidase and alkaline phosphatase, for example.
Gene cloning in
E. coli
vectors no longer requires a level of qualification needing several years experience as was still the case in the 1980s. Acquisition of these standard technologies often begins at school and is carried on in molecular biology courses at university in any country with a strong scientific tradition. Methodologies relating to DNA and RNA manipulation have been greatly simplified by the introduction of successive improvements designed by the entire scientific community and propagated through publications. Another decisive element that has led to simplification and also to reduction of the time needed to perform experiments relating to DNA and RNA has come from companies specialized in the supply of products for research into and use of microbiology and molecular biology. By supplying equipment that is ready-to-use and contains all the elements needed to perform even the most complex experimental operations in biology these companies have greatly contributed to make these technologies more accessible. These companies readily impart information on any technological improvement discovered in the world's scientific and medical laboratories to their biologist clients. To date none of them offer the products and the methodology that are the subject of this invention.
The different stages in the transformation of a bacterium or of eucaryotic microorganisms such as yeasts and filamentous fungi by a DNA vector are broadly as follows: recipient cells enabled to incorporate DNA by means of various treatments are put into contact with the DNA and are then put into culture in a liquid medium to allow the appearance of the selection property before being spread in Petri dishes containing a solid medium with the addition of the selection agent. At this stage a chromogenic or fluorescent substrate may also be added for the cloning or transfer vectors as a means to identify the colonies. Following one night or several days incubation, according to the growth rate of the organism concerned, those cells that initially recovered the vector and express the transgenes form colonies. These colonies are then recovered in order to prepare cultures, this time in liquid media containing the selection agent, in order to extract the extrachromosomal DNA which is then characterized on the basis of size, the presence of restriction enzyme sites, and possibly by sequencing. The volume of liquid culture depends on the quantity of DNA sought by the researcher, and may vary from a few milliliters to one liter or more. These technologies, as applied for instance to microorganisms such as
E. coli
and
S. cerevisiae
, are well known to those familiar with the art, and their degree of difficulty in no way limits implementation of the invention.
BACKGROUND OF THE INVENTION
The invention relates to a new method of preparing selective liquid and solid media for the selection and manipulation of recombinant microorganisms. The selective media are at present prepared in an identical way by all experimental biologists all over the world. The ingredients that compose a given medium are mixed with water in variously shaped containers usually made of glass and closed either with a cork ma
Armau Elise
Tiraby Gerard
Cayla
Larson & Taylor PLC
Naff David M.
Ware Deborah K.
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