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

Technology overview

E. I. du Pont de Nemours and Co. is a multinational company that conducts R&D in the science field including agriculture, electronics, health care, safety and protection.  The patent family described below discloses a method to detect sublethal levels of environmental contaminants using a lyophilized trangenic bacterium containing a luxCDABE gene complex that is under the control of a stress-inducible promoter.  Related scientific publications are:

  • Van Dyk et al. (1994).  Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions.  Appl Environ Microbiol. 60(5):1414-20.  (dnaKp::lux or grpEp::lux into E. coli)
  • Van Dyk et al. (1995).  Synergistic induction of the heat shock response in Escherichia coli by simultaneous treatment with chemical inducers.  J Bacteriol. 177(20):6001-4.  (lonp::lux into E. coli)
  • Van Dyk et al. (1995).  Responses to toxicants of an Escherichia coli strain carrying a uspA’::lux genetic fusion and an E. coli strain carrying a grpE’::lux fusion are similar.  Appl Environ Microbiol. 61(11):4124-7.  (uspAp::lux into E. coli)
  • Belkin et al. (1996).  Oxidative stress detection with Escherichia coli harboring a katG’::lux fusion.  Appl Environ Microbiol. 62(7):2252-6.  (katGp::lux into E. coli)
  • Vollmer et al. (1997).  Detection of DNA damage by use of Escherichia coli carrying recA’::luxuvrA’::lux, or alkA’::lux reporter plasmids.  Appl Environ Microbiol. 63(7):2566-71.  (recAp::luxuvrAp::lux, or alkAp::lux into E. coli)
  • Wagner and Van Dyk (1998).  Cryopreservation and reawakening.  Methods Mol Biol. 102:123-7.
  • Van Dyk (1998).  Stress detection using bioluminescent reporters of the heat-shock response.  Methods Mol Biol. 1998;102:153-60.  (review)
  • Bechor et al. (2002).  Recombinant microorganisms as environmental biosensors: pollutants detection by Escherichia coli bearing fabA’::lux fusions.  J Biotechnol. 94(1):125-32.  (fabAp::lux into E. coli)

Many of the transgenic E. coli containing an inducible promoter :: reporter gene construct are provided in the examples of WO 1994/13831, also filed by E. I. du Pont de Nemours and Co. (see chapter 5 – Detection of metals and other toxic compounds).

Details of patent documents

Patent or Publication No. Title, Independent Claims and Summary Assignee and licensing information
CA 2200702

  • Earlliest priority – 23 Nov 1994
  • Filed – 20 Nov 1995
  • Granted – 5 Dec 2000
  • Lapsed – 21 Nov 2005
  • Expected expiry – not applicable
Title – Lyophilized bioluminescent bacterial reagent for the detection of toxicants

Claim 1
A method of detecting the presence of a sublethal environmental insult with a lyophilized biological reagent, said reagent comprising

  • detector prokaryote microorganism containing an expressible luxCDABE gene complex under the control of a stress inducible promoter sequence, the method comprising the steps of:

(i) rehydrating the lyophilized biological reagent in a suitable amount of water wherein a baseline bioluminescence is produced;
(ii) immediately contacting the rehydrated reagent with a sample suspected of containing an environmental insult to form a reagent mixture;
(iii) incubating the mixture for at least 20 minutes and at a temperature of up to 30°C and;
(iv) detecting a change in bioluminescence from the mixture.

Claim 5
A lyophilized biological reagent comprising

  • a transformed bacteria containing an expressible luxCDABE gene complex under the control of a stress inducible promoter sequence.
Claim 6
A kit for detecting the presence of a sublethal environmental insult comprising the following in packaged combination:

(i) an aliquoted lyophilized biological reagent comprising;
(a) a detector prokaryotic microorganism cell containing a DNA fragment comprising a stress inducible promoter operably linked to the luxCDABE gene complex;
(b) a suitable buffer, and
(c) a cryoprotective reagent;
(ii) a rehydrating reagent; and
(iii) a suitable growth media.

The claims are generally drawn towards:

  • a method of detecting the presence of a sublethal environmental insult with a prokaryote microorganism containing an expressible luxCDABE gene complex under the control of a stress inducible promoter (claim 1)
  • a lyophilized biological reagent comprising a bacteria transformed with an expressible luxCDABEgene complex under the control of a stress inducible promoter sequence (claim 5)
  • a kit for detecting the presence of a sublethal environmental insult comprising a prokaryotic microorganism cell containing a DNA fragment comprising a stress inducible promoter operably linked to the luxCDABE gene complex (claim 6)

Definitions extracted from the description are:

  • (Environmental) insult – any substance or environmental change that results in an alteration of normal cellular metabolism in a bacterial cell or population of cells
  • Detector (prokaryote micro)organism – a prokaryote microorganism which contains a gene fusion consisting of a stress inducible promoter fused to the lux gene complex (luxCDABE gene complex) and which is capable of expressing the lux gene products in response to an environmental insult
  • Stress inducible promoter – any promoter capable of activating a stress gene and causing the expression of the stress gene product
  • Bioluminescence – phenomenon of light emission from any living organism
  • Lyophilized biological reagent – a detector organism which contains a gene fusion consisting of a stress inducible promoter fused to the lux gene complex and which is freeze-dried in a specific medium and is capable of expressing the lux gene products in response to an environmental insult, immediately upon rehydration
  • Operably linked – the fusion of two fragments of DNA in a proper orientation and reading frame to be transcribed into functional RNA.

Comments:

As this patent has lapsed due to non-payment of maintenance fees, this patent currently does not have any enforceable rights.

E. I. du Pont de Nemours and Company

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 793729

  • Earlliest priority – 23 Nov 1994
  • Filed – 20 Nov 1995
  • Granted – 21 Aug 2002
  • Expected expiry – 20 Nov 2015
Title – Lyophilized bioluminescent bacterial reagent for the detection of toxicants

Claim 1
A method of detecting the presence of an environmental insult with a lyophilized biological reagent said reagent comprising a detector organism containing an expressible lux gene complex under the control of a stress inducible promoter sequence, the method comprising the steps of:

(i) rehydrating the lyophilized biological reagent in a suitable amount of water wherein a baseline bioluminescence is produced;
(ii) immediately contacting the rehydrated reagent with a sample suspected of containing an environmental insult to form a reagent mixture;
(iii) incubating the mixture for at least 20 minutes and at a temperature of up to 300˚C and;
(iv) detecting a change in bioluminescence from the mixture.

Claim 10
A lyophilized biological reagent comprising a transformed bacteria containing an expressible luxgene complex under the control of a stress inducible promoter sequence.
Claim 11
A kit for detecting the presence of an environmental insult comprising the following in packaged combination:

(i) an aliquoted lyophilized biological reagent comprising;
(a) a detector cell containing a DNA fragment comprising a stress promoter gene operably linked to the lux gene complex;
(b) a suitable buffer; and
(c) a cryoprotective reagent;
(ii) a rehydrating reagent; and
(iii) a suitable growth media.

Designated contracting States at the time of grant are: Austria (patent lapsed as reported by INPADOC), Belgium, Switzerland, Germany, Denmark (patent lapsed as reported by INPADOC), Spain (patent lapsed as reported by INPADOC), France, United Kingdom, Greece (patent lapsed as reported by INPADOC), Ireland (patent lapsed as reported by INPADOC), Italy, Liechtenstein, Luxembourg (patent lapsed as reported by EPO), Monaco, Netherlands, Portugal (patent lapsed as reported by INPADOC), Sweden (patent lapsed as reported by INPADOC)

The claims are generally drawn towards:

  • a method of detecting the presence of a sublethal environmental insult with a detector organism containing an expressible lux gene complex under the control of a stress inducible promoter (claim 1)
  • a lyophilized biological reagent comprising a bacteria transformed with an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 10)
  • a kit for detecting the presence of a sublethal environmental insult comprising a detector cell containing a DNA fragment comprising a stress inducible promoter operably linked to the luxgene complex (claim 11)

Definitions extracted from the specification are provided in CA 2200702.

Comments:
There is no limit on the type of lux gene complex nor type of detector organism in EP 793729.

US 5731163

  • Earlliest priority – 23 Nov 1994
  • Filed – 22 Nov 1996
  • Granted – 24 March 1998
  • Expected expiry – 22 Nov 2016
Title – Lyophilized bioluminescent bacterial reagent for the detection of toxicants

Claim 1
A method of detecting the presence of a sublethal environmental insult with a lyophilized biological reagent said reagent comprising

  • a detector prokaryotic microorganism containing an expressible luxCDABE gene complex under the control of a stress inducible promoter sequence, the method comprising the steps of:

(i) rehydrating the lyophilized biological reagent in a suitable amount of water wherein a baseline bioluminescence is produced;
(ii) immediately contacting the rehydrated reagent with a sample suspected of containing an environment insult to form a reagent mixture;
(iii) incubating the mixture for at least 20 minutes and at a temperature of up to 30° C. and;
(iv) detecting an increase in bioluminescence from the mixture.

Claim 6
A lyophilized biological reagent comprising

  • bacteria transformed with an expressible luxCDABE gene complex under the control of a stress inducible promoter sequence.
Claim 7
A kit for detecting the presence of a sublethal environmental insult comprising the following in packaged combination:

(i) an aliquoted lyophilized biological reagent comprising;
(a) a detector prokaryotic microorganism cell containing a DNA fragment comprising a stress promoter gene operably linked to a luxCDABE gene complex;
(b) a suitable buffer; and
(c) a cryo protective reagent;
(ii) a rehydrating reagent; and
(iii) a suitable growth media.

The claims are generally drawn towards:

  • a method of detecting the presence of a sublethal environmental insult with a prokaryotic microorganism containing an expressible luxCDABE gene complex under the control of a stress inducible promoter (claim 1)
  • a lyophilized biological reagent comprising bacteria transformed with an expressible luxCDABEgene complex under the control of a stress inducible promoter sequence (claim 6)
  • a kit for detecting the presence of a sublethal environmental insult comprising a prokaryotic microorganism cell containing a DNA fragment comprising a stress inducible promoter operably linked to the luxCDABE gene complex (claim 7)

Definitions extracted from the specification are provided in CA 2200702.

Comments:

US 5731163 recites the same three independent claims as those of CA 2200702, which limits the detector organism to be a prokaryotic microorganism, and that the reporter gene is an expressible luxCDABE gene (and not any lux gene complex).

WO 1996/16187

  • Earlliest priority – 23 Nov 1994
  • Filed – 20 Nov 1995
  • Published – 30 May 1996
  • Expected expiry – not applicable
Title – Lyophilized bioluminescent bacterial reagent for the detection of toxicants

Claim 1
A method of detecting the presence of an environmental insult with a lyophilized biological reagent said reagent comprising a detector organism containing an expressible lux gene complex under the control of a stress inducible promoter sequence, the method comprising the steps of:

(i) rehydrating the lyophilized biological reagent in a suitable amount of water wherein a baseline bioluminescence is produced;
(ii) immediately contacting the rehydrated reagent with a sample suspected of containing an environmental insult to form a reagent mixture;
(iii) incubating the mixture for at least 20 minutes and at a temperature of up to 30˚C and;
(iv) detecting a change in bioluminescence from the mixture.

Claim 10
A lyophilized biological reagent comprising a transformed bacteria containing an expressible luxgene complex under the control of a stress inducible promoter sequence.
Claim 11
A kit for detecting the presence of an environmental insult comprising the following in packaged combination:

(i) an aliquoted lyophilized biological reagent comprising;
(a) a detector cell containing a DNA fragment comprising a stress promoter gene operably linked to the lux gene complex;
(b) a suitable buffer; and
(c) a cryoprotective reagent;
(ii) a rehydrating reagent; and
(iii) a suitable growth media.

The claims are generally drawn towards:

  • a method of detecting the presence of a sublethal environmental insult with a detector organism containing an expressible lux gene complex under the control of a stress inducible promoter (claim 1)
  • a lyophilized biological reagent comprising a bacteria transformed with an expressible lux gene complex under the control of a stress inducible promoter sequence (claim 10)
  • a kit for detecting the presence of a sublethal environmental insult comprising a detector cell containing a DNA fragment comprising a stress inducible promoter operably linked to the luxgene complex (claim 11)

Definitions extracted from the specification are provided in CA 2200702.

Comments:

Since this is a published application and not a granted patent, the claims above do not have enforceable rights.

Remarks National phase entry of WO 1996/16187 in Japan (JP H10/509049) is deemed to be withdrawn (notice of rejection created on 21 Oct 1999, last correspondence recorded on 18 Nov 1999).

Search strategy

Search details
Date of search 08/05/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms US ‘5731163’
Results 1
Comments This patent document was identified as a reference in the description of US 2001/034039 – Bioluminescent organism for detecting toxic substances