Patent applications filed and granted patents owned by E. I. du Pont de Nemours and Company 2

Technology overview

The patent family described in this section by E.I. du Pont de Nemours and Co. disclose a method to detect the presence of environmental insults (defined in the specification as ‘substance or environmental change that results in an alteration of normal cellular metabolism in a bacterial cell or population of cells’) by using a transgenic organism containing a luxgene complex that is under control of a stress-inducible promoter that is responsive to a regulatory circuit.  Examples in the patent specification provide the following constructs that were tested for luminescence upon exposure of transgenic E. coli strains containing the constructs to various chemical (e.g. ethanol, heavy metal compounds, hydrogen peroxide, mitomycin C) and physical (UV radiation) stressors:

  • grpEp::lux
  • lonp::lux
  • recAp::lux
  • uvrAp::lux
  • katGp::lux
  • micFp::lux
  • uspAp::lux
  • xthAp::lux
  • hisp::lux
  • lacp::lux
  • phoAp::lux – Transgenic E. coli responded to limiting phosphate in the medium
  • glnAp::lux – Transgenic E. coli responded to limiting nitrogen in the medium

Details of patent documents

Patent or Publication no. Title, Independent Claims and Summary Assignee and licensing information
CA 2150232

  • Earliest priority – 4 Dec 1992
  • Filed – 2 Dec 1993
  • Granted – 19 Dec 2000
  • Expected expiry – 2 Dec 2013
Title – A highly sensitive method for detecting environmental insults

Claim 1
A method of detecting the presence of an environmental insult comprising:(a) exposing a transformed detector E. coli to an environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating the presence of an environmental insult.
Claim 2
A method of detecting stress in a population of transformed E. coli comprising:(a) exposing a population of transformed detector E. coli to an environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating stress.
Claim 8
A transformed bioluminescent E coli capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult, the transformed bioluminescent E. coli comprising:(a) a stress inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) an expressible heterologous luxCDABE gone complex under the control of the stress inducible promoter sequence.
Claim 10
A method of detecting the presence of a environmental insult comprising:(a) exposing a transformed detector E. coli to a sublethal environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gone complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating the presence of an environmental insult.
Claim 11
E. coli selected from the group consisting of:(i) TV1076 having ATCC Number 69314 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a grpE stress inducible promoter sequence;
(ii) WM1302 having ATCC Number 69316 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a dnaK stress inducible promoter sequence;
(iii) TV1060 having ATCC Number 69142 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(iv) TV1061 having ATCC Number 69315 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(v) WM1021 having ATCC Number 69141 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence;
(vi) WM1026 having ATCC Number 69143 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence; and
(vii) WM1202 having ATCC Number 69313 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence.
Claim 12
A nucleic acid molecule, comprising:(a) a stress inducible promoter sequence wherein said promoter sequence is responsive to a regulatory circuit; and
(b) an expressible bacterial luxCDABE gene complex under control of said promoter sequence.

The claims are generally drawn towards:

  • a method of detecting the presence of an environmental insult comprising exposing E. coliengineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence (claim 1, 10)
  • a method of detecting stress in a population of transformed E. coli comprising exposing E. coliengineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence (claim 2)
  • a transformed bioluminescent E coli capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult (claim 8)
  • E. coli strains comprising an expressible heterologous lux gene complex under control of a stress inducible promoter sequence (claim 11)
  • a nucleic acid molecule comprising a stress inducible promoter sequence and an expressible bacterial luxCDABE gene complex (claim 12)

Definitions extracted from the specification are provided in US 5683868.

Comments:

The scope of the independent claims in granted CA 2150232 is the same as those of US 5683868.

E.I. du Pont de Nemours and Co.

1007 MARKET STREET WILMINGTON, DE 19898

Licensing information:
Ph +1-(781) 972-0607
Email dupont@yet2.com

DuPont has a website called ‘DuPont Technology Bank’, which contains information on licensing patented technology by their company:

http://dupont.t2h.yet2.com/t2h/page/homepage/

EP 673439 B1

EP 673439 B2

  • Earliest priority – 4 Dec 1992
  • Filed – 2 Dec 1993
  • Granted – 23 Apr 1997
  • Modified – 4 Jul 2001
  • Expected expiry – 2 Dec 2013
Title – Method for detecting environmental insults

Claim 1
A method of detecting environmental stress comprising:(a) exposing a detector organism to an environmental insult, said organism being genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit;and
(b) measuring a change in luminescence of said detector organism.
Claim 2
A method of detecting environmental stress comprising:(a) exposing a detector organism to a sublethal environmental insult, said organism being genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) measuring an increase in luminescence of said detector organism.
Claim 4
A method of detecting stress on a microorganism population, comprising:(a) exposing a detector organism to an environmental insult, said organism being genetically engineered to contain an expressible heterologous lux gene complex under the control of a stress inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) measuring a change in luminescence of said detector organism.
Claim 7
A transformed bioluminescent microorganism capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult, said microorganism comprising:(a) a stress inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) an expressible lux gene complex under the control of said stress inducible promoter sequence.
Claim 10
E. coli selected from the group consisting of:(i) TV1076 having ATCC Number 69314 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a grpE stress inducible promoter sequence;
(ii) WM1302 having ATCC Number 69316 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a dnaK stress inducible promoter sequence;
(iii) TV1060 having ATCC Number 69142 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(iv) TV1061 having ATCC Number 69315 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(v) WM1021 having ATCC Number 69141 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence;
(vi) WM1026 having ATCC Number 69143 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence; and
(vii) WM1202 having ATCC Number 69313 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence.
Claim 11
Nucleic acid molecules, comprising:(a) a stress inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) an expressible bacterial lux gene complex under control of said promoter sequence.

Note: amended phrases of the independent claims of EP 673439 B2 from EP 673439 B1 are indicated in bold.

The claims are generally drawn towards:

  • a method of detecting environmental stress comprising exposing a detector organism genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 1, 2)
  • a method of detecting stress on a microorganism population comprising exposing a detector organism genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 4)
  • a transformed bioluminescent microorganism capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult (claim 7)
  • E. coli strains comprising an expressible heterologous lux gene complex under control of a stress inducible promoter sequence (claim 10)
  • nucleic acid molecules comprising a stress inducible promoter sequence and an expressible bacterial lux gene complex (claim 11)

Definitions extracted from the specification are provided in US 5683868.

Comments:

All six independent claims in EP 673439 are relatively broader in scope compared to those of US 5683868 for the following two reasons:

  1. There is no limit on the type of detector organism.
  2. There is no limit on the type of lux genes included in the lux gene complex of the transgenic detector organism (minimum requirements of luxA and luxB are stated in the specification, see definitions for ‘lux‘ provided in US 5683868).

Designated contracting States at the time of grant (as of 4 Jul 2001) are: Austria, Belgium, Switzerland, Germany, Denmark, Spain, France, United Kingdom, Greece, Ireland, Italy, Liechtenstein, Luxembourg, Monaco, Netherlands, Portugal, Sweden

According to European Patent Register, VITO filed an opposition after the initial decision to grant on 23 Jan 1998 based on lack of novelty and insufficient disclosure (referred to their own PCT application WO 1992/15687 and other scientific publication as prior art), after which the claims were amended and the decision to maintain the patent in its amended form issued on 25 May 2001 (amended patent subsequently published on 4 Jul 2001).

US 5683868

  • Earliest priority – 4 Dec 1992
  • Filed – 2 Dec 1993 (check date)
  • Granted – 4 Nov 1997
  • Expected expiry – 4 Nov 2014
Title – Highly sensitive method for detecting environmental insults

Claim 1
A method of detecting the presence of an environmental insult comprising:(a) exposing a transformed detector E. coli to an environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating the presence of an environmental insult.
Claim 2
A method of detecting stress in a population of transformed E. coli comprising:(a) exposing a population of transformed detector E. coli to an environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a global regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating stress.
Claim 8
A transformed bioluminescent E coli capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult, the transformed bioluminescent E. coli comprising:(a) a stress inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) an expressible heterologous luxCDABE gene complex under the control the stress inducible promoter sequence.
Claim 10
A method of detecting the presence of a environmental insult comprising:(a) exposing a transformed detector E. coli to a sublethal environmental insult, the transformed detector E. coli being genetically engineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence wherein the promoter sequence is responsive to a regulatory circuit; and
(b) measuring an increase in luminescence of the transformed detector E. coli, the increase indicating the presence of an environmental insult.
Claim 11
E. coli selected from the group consisting of:(i) TV1076 having ATCC Number 69314 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a grpE stress inducible promoter sequence;
(ii) WM1302 having ATCC Number 69316 comprising a tolC- mutation and an expressible heterologous lux gene complex under the control of a dnaK stress inducible promoter sequence;
(iii) TV1060 having ATCC Number 69142 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(iv) TV1061 having ATCC Number 69315 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(v) WM1021 having ATCC Number 69141 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence;
(vi) WM1026 having ATCC Number 69143 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence; and
(vii) WM1202 having ATCC Number 69313 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence.
Claim 12
A nucleic acid molecule, comprising:
(a) a stress inducible promoter sequence wherein said promoter sequence is responsive to a regulatory circuit; and
(b) an expressible bacterial luxCDABE gene complex under control of said promoter sequence.

The claims are generally drawn towards:

  • a method of detecting the presence of an environmental insult comprising exposing E. coliengineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence (claim 1, 10)
  • a method of detecting stress in a population of transformed E. coli comprising exposing E. coliengineered to contain an expressible heterologous luxCDABE gene complex under the control of a stress-inducible promoter sequence (claim 2)
  • a transformed bioluminescent E. coli capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult (claim 8)
  • E. coli strains comprising an expressible heterologous lux gene complex under control of a stress inducible promoter sequence (claim 11)
  • a nucleic acid molecule comprising a stress inducible promoter sequence and an expressible bacterial luxCDABE gene complex (claim 12)

Definitions extracted from the specification are:

  • Promoter – a sequence of DNA, usually upstream of (5′ to) the protein coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at the correct site.
  • lux – the lux structural genes which include luxA, luxB, luxC, luxD and luxE and which are responsible for the phenomenon of bacterial bioluminescence. A lux gene complex might include all of the independent lux genes, acting in concert, or any subset of the lux structural genes so long as luxA and luxB are part of the complex.
  • Stress – the condition produced in a cell as the result of exposure to an environmental insult.
  • Environmental insult – any substance or environmental change that results in an alteration of normal cellular metabolism in a bacterial cell or population of cells.
  • Stress-inducible promoter – any promoter capable of activating a stress gene and causing increased expression of the stress gene product.
  • Detector organism (E. coli) – organism (E. coli) which contains a gene fusion consisting of a stress inducible promoter fused to a structural gene and which is capable of expressing the lux gene products in response to an environmental insult.
  • Regulatory circuit – there is no definition for this term. Table 1 in the specification lists the following circuits with corresponding stimuli, regulatory genes and responding genes: heat shock, SOS, H2O2, superoxide, fatty acid starvation, universal stress, resting state, stringent, catabolite activation, P utilization, and N utilization.
  • Subelethal level – levels below those needed to affect cell metabolism.

Comments:

Similar to the limitations added to the scope of the six independent claims granted in CA 2150232, claims granted in US 5683868 have the following limitations:

  1. The detector organism is limited to E. coli
  2. The lux gene complex must contain all five genes, luxAluxBluxCluxD and luxE.

According to the examples provided in the specification, tolC mutations of E. coli strains ATCC 69314 and ATCC 69316 in claim 11 confer inhanced permeability of organic compounds into the E. coli cells.

WO 1994/13831

  • Earliest priority – 4 Dec 1992
  • Filed – 2 Dec 1993
  • Published – 23 Jun 1994
  • Expected expiry – not applicable
Title – A highly sensitive method for detecting environmental insults

Claim 1
A method of detecting environmental stress comprising:(a) exposing a detector organism to an environmental insult, said organism being genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence; and
(b) measuring a change in luminescence of said detector organism.
Claim 2
A method of detecting environmental stress comprising:(a) exposing a detector organism to a sublethal environmental insult, said organism being genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence; and
(b) measuring an increase in luminescence of said detector organism.
Claim 4
A method of detecting stress on a microorganism population, comprising:(a)  exposing a detector organism to an environmental insult, said organism being genetically engineered to contain an expressible heterologous lux gene complex under the control of a stress inducible promoter sequence; and
(b)  measuring a change in luminescence of said detector organism.
Claim 7
A transformed bioluminescent microorganism capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult, said microorganism comprising:(a) a stress inducible promoter sequence; and
(b) an expressible lux gene complex under the control of said stress inducible promoter sequence.
Claim 10
E. coli selected from the group consisting of:(i) TV1076 having ATCC Number 69314 comprising a tolC mutation and an expressible heterologous lux gene complex under the control of a grpE stress inducible promoter sequence;
(ii) WM1302 having ATCC Number 69316 comprising a tolC mutation and an expressible heterologous lux gene complex under the control of a dnaK stress inducible promoter sequence;
(iii) TV1O6O having ATCC Number 69142 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(iv) TV1061 having ATCC Number 69315 comprising an expressible heterologous lux gene complex under control of a grpE stress inducible promoter sequence;
(v) W141021 having ATCC Number 69141 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence;
(vi) WM1026 having ATTC Number 69143 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence; and
(vii) Wb41202 having ATCC Number 69313 comprising an expressible heterologous lux gene complex under control of a dnaK stress inducible promoter sequence.
Claim 11
Nucleic acid molecules, comprising:(a) a stress inducible promoter sequence; and
(b) an expressible bacterial lux gene complex under control of said promoter sequence.

The claims are generally drawn towards:

  • a method of detecting environmental stress comprising exposing a detector organism genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 1, 2)
  • a method of detecting stress on a microorganism population comprising exposing a detector organism genetically engineered to contain an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 4)
  • a transformed bioluminescent microorganism capable of an increase in bioluminescence upon exposure to a sublethal level of environmental insult (claim 7)
  • E. coli strains comprising an expressible heterologous lux gene complex under control of a stress inducible promoter sequence (claim 10)
  • nucleic acid molecules comprising a stress inducible promoter sequence and an expressible bacterial lux gene complex (claim 11)

Definitions extracted from the specification are provided in US 5683868.

Comments:

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

Remarks
  1. National phase entry of WO 1994/13831 in Australia (AU 5730494) has lapsed on 25 Aug 1995.
  2. National phase entry of WO 1994/13831 in South Africa (ZA 9309078) was published as granted on 5 Jun 1995.

Search strategy

This patent was identified as reference to WO 1997/41251 titled ‘Recombinant nucleic acid sequences and methods for determining both genotoxicity and mutagenicity of a sample and the kinetics of the genetoxicity’ filed by VITO.