US 11,421,236 B1Granted

Biosensor for detecting TNT

AssigneeResearch Foundation of the City University of New York

InventorsChang-Hui Shen, Robert Corin

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Abstract

A biosensor for detecting nitrotoluenes. Two P. putida host populations (H-I and H-II) are engineered. H-1 undergoes fluorescence when a nitrotoluene is detected but it is also engineered to metabolize nitrotoluenes to toluene as its sole nitrogen-source. H-I is 1-ACC Deaminase inactive and is further engineered to efflux toluene and provide toluene to adjacent H-II. In H-II, ACC is the N-source and metabolizes toluene as the sole carbon and energy source available. The H-II cells are engineered to not be able to use medium fructose. The H-II population has a promoter/GFP construct with a promoter sensitive to toluene and thus they fluoresce from that first nitrotoluene metabolite i.e. toluene, produced by the H-I cells. This is achieved by making H-II cells mutants unable to transport and phosphorylate fructose i.e. PTSFRU gene knock-out.

Claims3

Dependent claims

A biosensor for detecting trinitrotoluene, the biosensor comprising:

a first Pseudomonas putida bacteria host (H-1) that is engineered to employ trinitrotoluene (TNT) as a sole N-source with toluene as a metabolic product, wherein the H-1 has been further engineered to comprise an inactivated endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, an Escherichia coli yqiF TNT-sensitive promoter linked to a green fluorescent protein (GFP), a chimeric DNA molecule with TNT sensitive promoter and fluorescent gene product;
and
a second Pseudomonas putida bacteria (H-2) that is engineered to employ toluene as a sole carbon source, wherein the H-2 has been further engineered to comprise an inactivated endogenous nitro reductase PnrA, PnrB and PnrC, a toluene-sensitive promoter from a TOL plasmid pWWO from Pseudomonas putida that is linked to a green fluorescent protein (GFP), a chimeric DNA molecule with toluene sensitive promoter and fluorescent gene product.

Dependencies are inferred from the claim text.

Facts

Application: 16/851,202Filed 17 Apr 2020 · us provisional application
Priority: 17 Apr 2019
Granted: 23 Aug 2022
Term ends: Not estimatedThe source records don’t support an estimate.

Classification22 codes

C12N 15/62
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: dNA sequences coding for fusion proteins
C07K 14/21
From bacteria: from Pseudomonadaceae
C07K 14/245
From Enterobacteriaceae , e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia: escherichia
C12N 9/0036
Acting on nitrogen containing compounds as donors: acting on NADH or NADPH