Microlife Technics, Inc.

Occidental Chemical Corporation

Microlife Genetics, Inc.

Ronald H Olsen

Peter A Vandenbergh

Joseph F Colaruotolo

Harold Rothchild

Cloning vectors are described which include the streptomycin resistance (Sm.sup.r) determinant derived from Tn904. A single site for the restriction endonuclease, AvaI, is present within the Tn904 determinant for Sm.sup.r. A method is described for preparing the Tn904 containing cloning vectors through transposition of Tn904 to a parent cloning vector and then cloning of the Sm.sup.r gene into another vector segment. The cloning vector is important for inserting deoxyribonucleic acid segments, which encode for various characteristics such as chemical production, antibiotic resistance or bacterial cell properties, in the Sm.sup.r gene AvaI cleaved site and which normally provides a marker for identification of transformed strains of bacteria.

Plasmid vectors including Tn904

An improved gene splicing and recombinant plasmid transformation method is described. The method includes mechanical fragmenting of chromosomal DNA followed by conventional digestion with a restriction enzyme and gene splicing into a vector to provide recombinant plasmids in a bank of at least about 100 different plasmids. The plasmids in the bank are provided for transformation into a suitable host, particularly a plasmid free bacterium of the same species from which the chromosomal DNA or the vector is derived. The method provides high transformation frequencies because of the presence of multiple 'super coiled' or closed coiled recombinant plasmids in the bank. The method also allows for the direct selection of many different phenotypic traits in a pool of the transformed hosts. The selected hosts are useful for the production of various gene products.

Gene splicing method and products produced therefrom

Improved cloning vectors derived from pRO1614 are described. One of these vectors, pRO1727, is suitable for cloning using DNA cleaved with the restriction endonuclease, PstI, and allows selection for the recovery of recombinant plasmids using tetracycline resistance. The cloning efficiency observed for pRO1727 is higher than described previously for pRO1614 and the host range of this vector is now restricted to Pseudomonas bacteria. Another vector, designated pRO1729, is described and developed from pRO1727 by deletion of a portion of its DNA and incorporation of a segment of DNA which encodes for resistance to the antibiotic, chloramphenicol. The chloramphenicol resistance determinant has a cleavage site for restriction endonuclease EcoRI within its chloramphenicol resistance determinant. Thus, DNA cloned into this site results in the loss of chloramphenicol resistance which can be detected subsequent to a cloning experiment. Both pRO1727 and pRO1729 are more useful in Pseudomonas for cloning than pRO1614.

Molecular cloning vectors for use in gram-negative bacteria

Selected new bacteria of the genus Pseudomonas, particularly the species Pseudomonas putida and Pseudomonas aeruginosa, which have the ability to utilize organic compounds from the generic groups aliphatic, cyclo aliphatic, aromatic and/or polynuclear aromatic hydrocarbons are described. The source of genetic materials facilitating degradation of the aromatic compounds are metabolic plasmids. In particular, Pseudomonas putida or other Pseudomonas obtained from soil samples and having a non-transmissible and stable ability to degrade hexane (as well as related aliphatic hydrocarbons) are used as starting strains to produce the new bacteria. Transconjugal mating and selection for these genetic traits resulted in the production of bacteria capable of utilizing representative compounds of all the generic groups of the previously listed organic compounds. The bacteria are useful for waste degradation.

Pseudomonas degradation of hydrocarbons

This invention relates to microbial methods and materials useful in the degradation of organic chemicals having toxic and obnoxious characteristics into innocuous materials compatible with the environment and to the process comprising identification, production and utilization of microorganisms for said purposes.

Microbial degradation of obnoxious organic wastes into innocuous

Microbial degradation of obnoxious organic wastes into innocucous

Compositions of selected strains of Pseudomonas bacteria having the ability to utilize halogenated aromatic compounds as a sole carbon source are described. The bacteria are isolated from environments where they have been in long association with halogenated aromatic compounds, usually analagous compounds. First L-tryptophan and then a halogenated aromatic hydrocarbon are used as sole carbon sources for isolating and testing the selected strains. The isolated Pseudomonas strains are Pseudomonas putida; Pseudomonas sp. NRRL-B-12,538 or NRRL-B-12,539 or transfer derivatives thereof and are useful for degrading halogenated aromatic pollutants, particularly mono- and di-chloroaromatics.

Pseudomonas compositions

The present invention relates to novel, broad bacterial host range small plasmid deoxyribonucleic acid rings which serve as cloning vehicles for DNA fragments, particularly those separated from other plasmid rings or from chromosones, recombined with the small plasmid rings and to the processes for recombining the plasmid rings and to processes for transferring them between host bacteria. In particular, the present invention relates to the aggregate plasmid ring RP1/pRO1600, to pRO1600 and plasmid ring derivatives thereof, particularly including pRO1601; pRO1613 and pRO1614, all of which are carried for reference purposes in Pseudomonas aeruginosa ATCC 15692 (also known as strain PAO1c) and are on deposit at the Northern Regional Research Laboratories (NRRL) of the United States Department of Agriculture at Peoria, Ill. The plasmid ring RP1 (also known as R1822) is deposited in Pseudomonas aeruginosa NRRL-B-12123 (and is a known plasmid ring). The pRO1600 portion of the aggregate is a new plasmid ring. The novel small plasmid rings are particularly useful for recombinant genetic manipulation wherein the DNA fragments are introduced into the plasmid rings to produce useful, cloneable characteristics in the host bacterium, particularly chemical generating characteristics.

DNA Fragments for forming plasmids

The present invention relates to novel, broad bacterial host range small plasmid deoxyribonucleic acid rings which serve as cloning vehicles for DNA fragments, particularly those separated from other plasmid rings or from chromosones, recombined with the small plasmid rings and to the processes for recombining the plasmid rings and to processes for transferring them between host bacteria. In particular, the present invention relates to the aggregate plasmid ring RP1/pRO1600, to pRO1600 and plasmid ring derivatives thereof, particularly including pRO1601; pRO1613 and pRO1614, all of which are carried for reference purposes in Pseudomonas aeruginosa ATCC 15692 (also known as strain PAO1c) and are on deposit at the Northern Regional Research Laboratories (NRRL) of the U.S. Department of Agriculture at Peoria, Il. The plasmid ring RP1 (also known as R1822) is deposited in Pseudomonas aeruginosa NRRL-B-12123 (and is a known plasmid ring). The pRO1600 portion of the aggregate is a new plasmid ring. The novel small plasmid rings are particularly useful for recombinant genetic manipulation wherein the DNA fragments are introduced into the plasmid rings to produce useful, cloneable characteristics in the host bacterium, particularly chemical generating characteristics.

Process for preparing broad host range small plasmid rings as cloning

The present invention relates to novel, broad bacterial host range small plasmid deoxyribonucleic acid rings which serve as cloning vehicles for DNA fragments, particularly those separated from other plasmid rings or from chromosones, recombined with the small plasmid rings and to the processes for recombining the plasmid rings and to processes for transferring them between host bacteria. In particular, the present invention relates to the aggregate plasmid ring RP1/pRO1600, to pRO1600 and plasmid ring derivatives thereof, particularly including pRO1601; pRO1613 and pRO1614, all of which are carried for reference purposes in Pseudomonas aeruginosa ATCC 15692 (also known as strain PAO1c) and are on deposit at the Northern Regional Research Laboratories (NRRL) of the U. S. Dept. of Agriculture at Peoria, Ill. The plasmid ring RP1 (also known as R1822) is deposited in Pseudomonas aeruginosa NRRL-B-12123 (and is a known plasmid ring). The pRO1600 portion of the aggregate is a new plasmid ring. The novel small plasmid rings are particularly useful for recombinant genetic manipulation wherein the DNA fragments are introduced into the plasmid rings to produce useful, cloneable characteristics in the host bacterium, particularly chemical generating characteristics.

Broad host range small plasmid rings as cloning vehicles

A frozen stabilized bacterial product consisting of Pediococcus cerevisiae mixed with a stabilizing agent, such as glycerol, and a nutrient medium. A process of making a fermented meat product in which the thawed concentrate is mixed with a meat formulation in an amount of at least .1 percent by weight, then the mixture is heated to between 110.degree. and 125.degree.F. for from 8 to 15 hours and then the bacterial action is terminated.

Growing community of inventors

Ann Arbor, MI, United States of America

Ronald H Olsen