Patent applications filed and patents owned by Bio-Orbit Oy

Technology overview

Dr Matti Korpela and Dr Matti Karp (Department of Biochemistry, University of Turku at the time of filing) developed a recombinant DNA plasmid, the replication of which could be regulated by an exogenous stimulant by subjecting a constitutive promoter under the control of a repressor gene (high temperature is provided in the examples in the patent document) and contained a luciferase gene.  This plasmid was introduced into E. coli, and was used as a reporter that detects the presence of agents (e.g. cadmium, antibiotics, UV light) that in any way affect the cell machinery by introducing a sample containing an agent, inducing plasmid replication with the stimulant, and detecting the level of decrease in light emission.  The related non-patent publication of this technology is Korpela M and Karp M (1988).  Stable light-producing Escherichia coli.  Biotechnol Lett. 10(6):383-388.

Bio-Orbit Oy was a company based in Helsinki that developed and distributed luminometers and related reagents.  This company was later bought by Thermo LabSystems Oy in 1999 (which is a subsidiary of Thermo Electron Corp., MA, USA).

Details of patent documents

Patent or publication no. Title, Independent Claims and Summary Assignee and licensing information
EP 469021

  • Earliest priority – 20 Apr 1989
  • Filed – 20 Apr 1990
  • Granted – 15 Nov 1995
  • Expected expiry – 20 Apr 2010
Title – Determination of factors affecting gene regulation and/or gene replication

Claim 1
A method for determining the presence or amount of a factor in a sample to be tested, wherein the factor affects directly or indirectly the DNA, RNA and/or proteins of the cell or the synthesis mechanisms thereof, characterized ina) incubating a sample to be tested with a population of transformed cells for a period sufficient to allow said factor, if present in the sample, to affect said cells, said cells being transformed with a recombinant-DNA plasmid, the replication of said plasmid being subject to a regulatable promoter, which can be induced by an exogenous stimulus independent of replication of the cells;
b) then applying said exogenous stimulus to said cells to induce replication of said plasmid, whereupon the copy number of the plasmid will begin to increase in the cell, if the factor has not affected the plasmid by inhibiting the replication; and
c) the change in the copy number of the recombinant-DNA plasmid in the cell is determined directly or indirectly, and compared with a reference test in which the factor was not present or in which it was present in a known amount, and thereby the presence or the amount of the factor is determined.
Claim 8
A method for determining the presence or amount of a factor in a sample to be tested, wherein the factor affects directly or indirectly the DNA, RNA and/or proteins of the cell or the synthesis mechanisms thereof, characterized ina) incubatiang a sample to be tested with a population of transformed cells for a period sufficient to allow said factor, if present in the sample, to affect said cells, said cells being transformed with a high copy number recombinant DNA plasmid containing a DNA sequence encoding a marker protein or a part thereof essential to the biological activity of said protein, said sequence being coupled to a regulatable promoter such that expression of said marker protein is inducible by an exogenous stimulus;
b) then applying said exogenous stimulus to said population of cells to induce expression of said marker protein, whereby the amount of the marker protein in the cell starts to grow, if the factor has not affected the protein synthesis directly or indirectly;
c) determining the amount of the marker protein expressed by said population of cells, and comparing said amount with a reference test in which the factor was not present or in which it was present in a known amount, and thereby the presence or the amount of the factor is determined.

The claims are generally drawn towards:

  • a method for determining the presence or amount of a factor that affects directly or indirectly the DNA, RNA and/or proteins of the cell or the synthesis mechanisms thereof (claim 1, 8)

Definitions extracted from the specification are provided in WO 1990/12887.

Comments:

Designated contracting States at the time of grant are Germany (lapsed as reported by INPADOC), Spain, France, United Kingdom, Italy, Netherlands.

The two independent claims introduced above in EP 469021 have been granted without substantial limitations being added from WO 1990/12887.

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US 6475719

  • Earliest priority – 20 Apr 1989
  • Filed – 21 Oct 1991
  • Granted – 5 Nov 2002
  • Expected expiry – 5 Nov 2022
Title – Determination of factors affecting gene regulation and/or gene replication

Claim 1
A method for determining the presence or amount of at least one inhibitory factor in the sample to be tested, wherein the inhibitory factor inhibits one or more of DNA synthesis, transcription of DNA, translation of RNA, cell wall synthesis, cell membrane function or metabolic functions which participate in or influence these processes, said method comprising:a) incubating a sample to be tested with a population of transformed cells for a period sufficient to allow an inhibitory factor, if present in the sample, to affect said cells, said cells being transformed with a recombinant DNA plasmid, and replication of said plasmid being inducible by an exogenous stimulus independent of replication of the cells and further wherein said cells are sensitive to inhibition by the inhibitory factor;
b) then applying said exogenous stimulus to said cells in an amount sufficient to induce replication of said plasmid in the absence of the inhibitory factor; and
c) determining the amount of DNA produced by said population of cells, the amount of the inhibitory factor in said sample being correlated with a reduction in the amount of DNA produced compared to the amount of DNA produced by a like cell population by application of a like amount of exogenous stimulus in the absence of said sample.
Claim 10
A method for determining the presence or amount of at least one inhibitory factor in the sample to be tested, wherein the inhibitory factor inhibits one of more of DNA synthesis, transcription of DNA, translation of RNA, cell wall synthesis, cell membrane function or metabolic functions which participate in or influence these processes, said method comprising:a) incubating a sample to be tested with a population of transformed cells for a period sufficient to allow the inhibitory factor, if present in the sample, to affect said cells, said cells being transformed with a high copy number recombinant DNA plasmid containing a sequence encoding a marker protein which is detectable when expressed, said sequence being coupled to a regulatable promoter such that expression of said marker protein is inducible by an exogenous stimulus;
b) then applying said exogenous stimulus to said population of cells in an amount sufficient to induce expression of said marker protein in the absence of the inhibitory factor; and
c) determining the amount of said marker protein expressed by said population of cells, the amount of the inhibitory factor in the sample being correlated with a reduction in the amount of said marker protein produced compared to the amount of said marker protein produced by a like cell population by application of a like amount of exogenous stimulus in the absence of the sample.
Claim 27
A method for determining the presence of at least one inhibitory factor selected from the group consisting of ampicillin, chloramphenicol, oxytetracycline, streptomycin, erythromycin, ofloxacin, ciprofloxacin, actinomycin and trimethoprim in a sample of a biological fluid selected from the group consisting of blood, plasma, serum, urine, semen and milk, said method comprising:a) obtaining a population of cells transformed with a high copy number recombinant DNA plasmid containing a sequence encoding luciferase, wherein said sequence is coupled to a regulatable promoter such that expression of said sequence is inducible by application of a chemical inducer to cells containing said plasmid;
b) incubating said sample with said cell population in a medium suitable for growth of said cells for a period sufficient to allow said inhibitory factor if present to affect said cells;
c) then applying said chemical inducer to said cells in an amount which in the absence of said inhibitory factor is sufficient to induce expression of said sequence; and
d) determining the amount of luciferase expressed by said cell population, the amount of said inhibitory factor in said sample being correlated with the reduction in the amount of luciferase produced relative to the amount of luciferase produced by a like cell population by application of a like amount of said chemical inducer in the absence of said sample.

The claims are generally drawn towards:

  • a method for determining the presence or amount of at least one inhibitory factor that inhibits one or more of DNA synthesis, transcription of DNA, translation of RNA, cell wall synthesis, cell membrane function or metabolic functions which participate in or influence these processes (claim 1, 10)
  • a method for determining the presence of at least one inhibitory factor (selected from the group consisting of eight entibiotics) in a sample of a biological fluid (selected from the group consisting of six fluids) (claim 27)

Definitions extracted from the specification are provided in WO 1990/12887.

Comments:

The main limitation that has been added to the granted independent claims introduced above is that the reporting signal in response to an “inhibitory factor” must be “a reduction in the amount of DNA produced” with cells exposed to an inhibitory factor, when compared with cells without such exposure.

WO 1990/12887

  • Earliest priority – 20 Apr 1989
  • Filed – 20 Apr 1990
  • Claims amended – 17 Sept 1990
  • Published – 1 Nov 1990
  • Expected expiry – not applicable
Title – Determination of factors affecting gene regulation and/or gene replication

Claim 1
A method for determining a factor affecting a cell, the factor affecting the DNA, the RNA and/or proteins of the cell or the synthesis machineries thereof, characterized in thata) into the cell is transferred a recombinant DNA plasmid, in which the starting point or points of the machinery responsible for the replication are subject to a regulatable promoter, which can be controlled either by a positive or a negative feedback;
b) the cell containing the recombinant DNA plasmid is brought into contact with the affecting factor;
c) the affecting factor is allowed to affect the cell containing the recombinant DNA plasmid for a suitable time, and subsequently the promoter regulating the starting point of the machinery responsible for the replication of the recombinant DNA plasmid is activated, whereby the copy number of the plasmid starts growing in the cell, unless the affecting factor has not affected the plasmid in a manner inhibiting the replication;
d) the shift of the copy number of the recombinant DNA plasmid is determined directly or indirectly.
Claim 8
A method for determining a factor affecting the cell, the factor affecting directly or indirecty the DNA, RNA and/or proteins of the cell or their synthesis machineries, characterized in thata) to the cell is transferred a recombinant DNA plasmid, present in many copies in the cell and the plasmid contains as a marker protein a protein of a virus, a procaryotic cell or an eucaryotic cell or a DNA sequence encoding a part essential with regard to its biological activity, the expression of which is subject to a regulatable promoter and is controlled either by a negative or a positive feedback;
b) the cell containing the recombinant DNA plasmid is brought into contact with the affecting factor;
c) the affecting factor is allowed to affect the cell containing the recombinant DNA plasmid for a suitable time, after which the regulatable promoter controlling the expression of the marker protein of the recombinant DNA plasmid is activated, whereby the amount of marker protein in the cell starts to grow, unless the affecting factor has affected the protein synthesis directly or indirectly;
d) the change of the amount/activity of the marker protein encoded by the recombinant DNA plasmid is measured.

The claims are generally drawn towards:

  • a method for determining the presence or amount of a factor that affects directly or indirectly the DNA, RNA and/or proteins of the cell or the synthesis mechanisms thereof (claim 1, 8)

Definitions extracted from the specification are:

  • Cell – procaryotic and eucaryotic organisms
  • Regulatable promoter – there is no definition for this term, therefore there is no apparent limitation on the type of regulatable promoter that can be used.  A subsequent dependent claim recites the following promoter sequences: “a strong promoter selected from the group consisting of lambda PL and PR promoters, lac, trp, and hybrid tac promoters”.  The term ‘regulatable promoter’ may be different to the definition of ‘inducible promoter’ that is used in other bioindicator systems, as the ‘regulatable promoter’ in this patent application is a constitutive promoter that is repressed by a repressor gene, whereas an ‘inducible promoter’ is a non-constitutive promoter that is directly induced when a stimulant is present.
  • Many copies – there is no definition for this term. Examples of plasmid copy numbers that are provided in the specification are:
    • pCSS112 (ori under control of the PL promoter from lambda phage) – 60
    • pCSS108 (ori under control of the lac promoter from E. coli) – 600
  • Marker protein – there is no limitation on the type of marker protein that can be used.  A subsequent dependent claim recites the following genes: “selected from the group consisting of alpha-amylase, alkaline phosphatase, β-galactosidase, luciferase, peroxidase, T4 lysozyme, β-glucuronidase, oxidoreductase and pyrophosphatase”.

Comments:

Since this is a published application and not a granted patent, currently there are no enforceable rights.

The term ‘regulatable promoter’ is different to the definition of ‘inducible promoter’ that is used in other bioindicator systems, as the ‘regulatable promoter’ in this patent application is a constitutive promoter that is repressed by a repressor gene, whereas an ‘inducible promoter’ is a non-constitutive promoter that is directly induced when a stimulant is present.

Remarks Two related patents have been granted in Finland (FI 891899, granted 31 Aug 2004; FI 88309, granted 31 Aug 2004).

Search strategy

Search details
Date of search 03/07/2006
Database searched Patent Lens
Type of search Structured, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms cadmium AND (Korpela in inventor)
Results 3
Comments Of the 3 results identified using these search terms, one result was identified as being of particular interest based on their abstracts and a review of their claims.