Patent applications filed and patents owned by VITO

Technology overview

VITO (Vlaamse instelling voor technologisch onderzoek, Flemish Institute for Technological Research) is a research organization in Belgium with a focus on contract research in the fields of energy, environment and materials technology.  The patent family described below by VITO discloses a fused gene that contains an inducible promoter in the presence of metals and/or xenobiotic compounds, and a luxCDABE operon as the reporter gene.  Processes for producing transgenic Escherichia coli containing the fused gene are also claimed.  Dr Philippe Corbisier and co-researchers used pMOL28 and pMOL30 (megaplasmids from Alcaligenes eutrophus CH34 that confer resistance to several heavy metals) to isolate metal-indicuble promoters, fuse them to luxCDABE and introduced the gene fusion into E. coli.  Other E. coli biosensors specific for chromium, nickel, copper and biphenyl detection were created via conjugation of luxCDABE-containing E. coli with particular A. eutrophus strains (SV661 for chromium, AE453 for nickel, DS185 or DS310 for copper, and A5 for biphenyl).  PCT application WO 1992/15687 is cited in a review by van der Lelie et al. (1994).  The use of biosensors for environmental monitoring.  Res. Microbiol. 145(1):67-74, and granted US patent US 5786162 is cited in a review by Kohler et al. (2000).  Reporter gene bioassays in environmental analysis. Fresenius J. Anal. Chem. 366(6-7):769-79.

Details of patent documents

Patent or Publication no.

Title, Independent Claims and Summary

Assignee and licensing information

CA 2105172

  • Earliest priority – 1 Mar 1991
  • Filed – 28 Feb 1992
  • Granted – 15 Oct 2002
  • Expected expiry – 28 Feb 2012
Title – Fused genes and their use for determining the presence of metals or of xenobiotic compounds

Claim 1
A fused gene comprisinga promoter sequence derived from a gene of Alcaligenes eutrophus strain CH34, SV661, DS185, AE453, or A5, said gene being a regulatory gene involved in the expression of either the resistance to one or several metals or the catabolism of one or several xenobiotic compounds, said promoter from said regulatory gene being an inducible promoter and is inducible in the presence of said metals or xenobiotic compounds, or both, and

downstream of the promoter, a five gene lux (CDABE) operon said genes coding for subunit .alpha. and .beta. of luciferase, a fatty acid reductase, an acyltransferase and an acylprotein synthase, said operon being under the operational control of said promoter, wherein at least one of said genes produces a detectable signal.

Claim 17
A process for preparing a bacterial cellular host wherein said bacterial cellular host emits light in the presence of zinc with a detection limit of 1 ppm and dynamic ravage between 1 and 23 ppm zinc comprising the steps of:(a) digesting with SalI a plasmid pBR325 comprising a czc fragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34 and surrounding EcoRI fragment to obtain a promoter and a gene encoding resistance to zinc;
(b) inserting said promoter and said gene encoding resistance to zinc of step
(a) into a plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M., Institut Pasteur, rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, under No. I-1050), at its multiple cloning site, and comprising the lux operon of Vibrio fischeri, to obtain a replicable plasmid;
(c) transforming the replicable plasmid of (b) in E. coli;
(d) selecting the inserted plasmid on ampicillin plates with various concentrations of zinc; and
(e) detecting the light producing E. coli in the presence of zinc.
Claim 18
A process for preparing a bacterial cellular host wherein said bacterial cellular host emits light in the presence of cobalt with a detection limit of 1 ppm and dynamic range between 1 and 23 ppm cobalt comprising the steps of:(a) digesting with SalI a plasmid pBR325 comprising a czc fragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34 and surrounding EcoRI fragment to obtain a promoter and a gene encoding resistance to cobalt;
(b) inserting said promoter and said gene encoding resistance to cobalt of step (a) into a plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M. Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, under No. I-1050), at its multiple cloning site, and comprising the lux operon of Vibrio fischeri, to obtain a replicable plasmid;
(c) transforming the replicable plasmid of (b) in E. coli;
(d) selecting the inserted plasmid on ampicillin plates with various concentrations of cobalt; and
(e) detecting the light producing E. coli in the presence of cobalt.

The claims are generally drawn towards:

  • a fused gene comprising an inducible promoter by metals or xenobiotic compounds, or both, and a lux (CDABE) operon (claim 1)
  • a process for preparing a bacterial cellular host that emits light in the presence of zinc (claim 17)
  • a process for preparing a bacterial cellular host that emits light in the presence of cobalt (claim 18)

Definitions extracted from the description are:

  • metal – (one designates) the transition metals, the rare earth, the elements having metallic properties in the families lila, IVa, Va and VIa of the Mendelieff table
  • (said) operon being under the operational control of (said) promoter – the promoter of the gene producing a detectable signal has been deleted
  • inducible (promoter) in the presence of said metals – there is a minimum concentration of said metal under which the promoter is not induced
  • lux (CDABE) – (in alphabetical order of the genes) genes coding for subunit alpha and beta of luciferase, a fatty acid reductase, an acyltransferase and an acylprotein synthase… can originate from Vibrio fischeri or from Vibrio harveyi or from Photobacterium phosphoreum or from Xenorhabdus luminescens

Comments:

Claim 1 recites a fused gene comprising an inducible promoter by metals or xenobiotic compounds, or both, and a lux (CDABE) operon, which can be used to transform any host organism. However, the origin of the inducible promoter is limited to those of Alcaligenes eutrophus strain CH34, SV661, DS185, AE453, or A5, which implies that a promoter that has the same property but originates from an organism other than the Alcaligenes eutrophus strains mentioned in claim 1 is not protected against use in this particular patent.

VITO

BOERETANG 200
B-2400
MOL BELGIUM

Ph + 32 14 33 55 11
Fax + 32 14 33 55 99

vito@vito.be

EP 573500 B1

EP 573500 B2

  • Earliest priority – 1 Mar 1991
  • Filed – 28 Feb 1992
  • Granted – 22 April 1998
  • Modified – 2 May 2002
  • Expected expiry – 28 Feb 2012
Title – Fused genes and their use for determining the presence of metals or of xenobiotic compounds

Claim 1
Fused gene containing:-  a promoter sequence obtainable from (a) gene(s) of Alcaligenes eutrophus and encoding either the resistance to one or several metal(s) or the catabolism of one or several xenobiotic compound(s), said promoter being inducible in the presence of said metal(s) or xenobiotic compound(s), or both,
– and downstream of the promoter, a five gene IuxCDABE operon, said genes coding respectively for a fatty acid reductase, an acyl transferase, two subunits Q and 3 of the luciferase and an acylprotein synthase, said lux operon being under the operational control of said Alcaligenes eutrophus promoter.
Claim 15
Transconjugants forming biosensors emitting light in the presence of metals or xenobiotics characterized in that they are obtained by the conjugation of Alcaligenes eutrophus and E. coli, wherein- Alcaligenes eutrophus contains a gene encoding the resistance to a metal or encoding the catabolism of a xenobiotic compound, and possibly a marker of the presence of the gene,
–   E. coli contains a transposon containing the luxCDABE operon of Vibrio fischeri without its own promoter and possibly a marker of the presence of said gene.

Designated contracting States at the time of grant are: Austria (AT 165394), Belgium, Switzerland, Germany (DE 69225221), Denmark (patent lapsed as reported on EPO), Spain (ES 2117665), France, United Kingdom, Greece (patent lapsed as reported on EPO), Italy, Liechtenstein, Luxembourg, Monaco (patent lapsed as reported on INPADOC), Netherlands, Sweden

The claims are generally drawn towards:

  • a fused gene containing an inducible promoter by metals or xenobiotic compounds, and a luxCDABE operon (claim 1)
  • transconjugants forming biosensors emitting light in the presence of metals or xenobiotics by the conjugation of Alcaligenes eutrophusand E. coli (claim 15)

Definitions extracted from the specification are provided in CA 2105172.

Comments:

Claim 1 of EP 573500 does not have a limit on the type of Alcaligenes eutrophus strain from which the inducible promoter is obtained.

US 5786162

  • Earliest priority – 1 Mar 1991
  • Filed – 28 Feb 1992
  • Granted – 28 Jul 1998
  • Expected expiry – 28 Jul 2015
Title – Fused genes and their use for determining the presence of metals or of xenobiotic compounds

Claim 1
A fused gene comprisinga promoter sequence derived from a gene of Alcaligenes eutrophus strain CH34, SV661, DS185, AE453, or A5, said gene being a regulatory gene involved in the expression of either the resistance to one or several metals or the catabolism of one or several xenobiotic compounds, said promoter from said regulatory gene being an inducible promoter and is inducible in the presence of said metals or xenobiotic compounds, or both, and

downstream of the promoter, a five gene lux (CDABE) operon said genes coding for subunit α and β of luciferase, a fatty acid reductase, an acyltransferase and an acylprotein synthase, said operon being under the operational control of said promoter, wherein at least one of said genes produces a detectable signal.

Claim 17
A process for preparing a bacterial cellular host wherein said bacterial cellular host emits light in the presence of zinc with a detection limit of 1 ppm and dynamic range between 1 and 23 ppm zinc comprising the steps of:(a) digesting with SalI a plasmid pBR325 comprising a czc fragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34 and surrounding EcoRI fragment to obtain a promoter and a gene encoding resistance to zinc;

(b) inserting said promoter and said gene encoding resistance to zinc of step (a) into a plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M., Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, under No. I-1050), at its multiple cloning site, and comprising the lux operon of Vibrio fischeri, to obtain a replicable plasmid;

(c) transforming the replicable plasmid of (b) in E. coli;

(d) selecting the inserted plasmid on ampicillin plates with various concentrations of zinc; and

(e) detecting the light producing E. coli in the presence of zinc.

Claim 18
A process for preparing a bacterial cellular host wherein said bacterial cellular host emits light in the presence of cobalt with a detection limit of 1 ppm and dynamic range between 1 and 23 ppm cobalt comprising the steps of:(a) digesting with SalI a plasmid pBR325 comprising a czc fragment of plasmid pMOL30 from Alcaligenes eutrophus strain CH34 and surrounding EcoRI fragment to obtain a promoter and a gene encoding resistance to cobalt;
(b) inserting said promoter and said gene encoding resistance to cobalt of step (a) into a plasmid pUCD615 (in E. coli CM600 deposited at the C.N.C.M. Institut Pasteur, 28 rue du Docteur Roux, 75015 Paris, on Feb. 28, 1991, under No. I-1050), at its multiple cloning site, and comprising the lux operon of Vibrio fischeri, to obtain a replicable plasmid;
(c) transforming the replicable plasmid of (b) in E. coli;
(d) selecting the inserted plasmid on ampicillin plates with various concentrations of cobalt; and
(e) detecting the light producing E. coli in the presence of cobalt.

The claims are generally drawn towards:

  • a fused gene comprising an inducible promoter by metals or xenobiotic compounds, or both, and a lux (CDABE) operon (claim 1)
  • a process for preparing an E. coli host that emits light in the presence of zinc (claim 17)
  • a process for preparing an E. coli host that emits light in the presence of cobalt (claim 18)

Definitions extracted from the specification are provided in CA 2105172.

Comments:

The three independent claims in US 5786162 are the same as those of CA 2105172, both of which are narrower than the two independent claims of EP 573500 in terms of the limit on the type of Alcaligenes eutrophus strain from which the inducible promoter is obtained, and the type of metals that the recombinant E. coli can detect (zinc and cobalt).

WO 1992/15687

  • Earliest priority – 1 Mar 1991
  • Filed – 28 Feb 1992
  • Published – 17 Sept 1992
  • Expected expiry – not applicable
Title – Fused genes and their use for determining the presence of metals or of xenobiotic compounds

Claim 1
Fused gene containing :- the promoter sequence of (a) gene(s) encoding the resistance to one or several metal(s) or encoding the catabolism of one or several xenobiotic compound(s), said promoter being inducible in the presence of said metal(s) or xenobiotic compound(s), or both,

– and downstream the promoter, a gene producing a detectable signal such as light emitting gene, said gene being under the control of said promoter, said gene producing a detectable signal being located at a position such that the induction of the promoter causes the transcription of the gene producing a detectable signal and such that there is no terminator between the promoter and the gene producing a detectable signal, said gene being such that it enables to recycle fatty acid (which has been generated during the reaction responsible for the detectable signal) into aldehyde.

The claims are generally drawn towards:

  • a fused gene containing an inducible promoter by metals or xenobiotic compounds or both, and a gene producing a detectable signal (claim 1)

Definitions extracted from the specification are provided in CA 2105172.

Comments:

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

Search strategy

Search details
Date of search 17/05/2006
Database searched Patent Lens
Type of search Simple, stemming on
Collections searched AU-B, US-A, US-B, EP-B, WO
Search terms VITO in applicant
Results 3
Comments Of the 3 results identified using these search terms, 3 results were identified as being of particular interest based on their abstracts and a review of their claims.