Bruce, Neil Charles (University of Cambridge Institute of Biotechnology Tennis Court Road Cambridge CB2 1QT, GB)
French, Christopher Edward (Institute of Cellular & Molecular Biology University of Edinburgh Darwin Building Mayfield Road Edinburgh, EH9 3JR, GB)
Nicklin, Stephen (Defence Evaluation & Research Agency Ively Road Farnborough Hampshire GU14 0LX, GB)
Bruce, Neil Charles (University of Cambridge Institute of Biotechnology Tennis Court Road Cambridge CB2 1QT, GB)
French, Christopher Edward (Institute of Cellular & Molecular Biology University of Edinburgh Darwin Building Mayfield Road Edinburgh, EH9 3JR, GB)
| 1. | An Enterobacter cloacae bacterial strain referred to as"PB2"and deposited as NCIMB 40718, and mutants and variants thereof, for use in the biodegradation of TNT. |
| 2. | The use of bacterial strain E. cloacae PB2 of claim 1 in the preparation of a composition used for the biodegradation of TNT in an environment. |
| 3. | A method for the biodegradation of TNT in an environment comprising the steps of inoculating the environment with a sample of bacterial isolate E. cloacae PB2 of claim 1 and allowing the isolate to degrade the TNT in the environment. |
| 4. | A method according to claim 3 wherein TNT and PETN in the environment are degraded by the isolate. |
| 5. | A PETN reductase enzyme having the amino acid sequence shown in SEQ ID NO: 1 or a derivative thereof for use in the biodegradation of TNT. |
| 6. | The use of PETN reductase enzyme of claim 5 in the preparation of a composition used for the biodegradation of TNT in an environment. |
| 7. | A method for the biodegradation of TNT in an environment comprising the steps of adding to the environment a quantity of PETN reductase enzyme of claim 5 and maintaining the mixture under conditions appropriate for degradation of the contaminant by PETN reductase enzyme. |
| 8. | A kit for biodegradation of an environment contaminated with TNT comprising a store of PETN reductase of claim 5 and a means of contacting the environment with PETN reductase in the presence of NADPH and maintaining the environment under conditions appropriate for the degradation of TNT by PETN reductase. |
TNT has been manufactured in large quantities for use in munitions. Manufacture, storage, testing, use and disposa of such munitions have resulted in the contamination of large amounts of soil and water with TNT and related compound. Further such contamination is likely to occur in the future. TNT is highly recalcitrant to biodegradation and as a result contamination has persiste in the environment (Rosenblatt et al, 1991, 'Organic explosives and related compounds', pp 195-234,'Handbook of Environmental Chemistry', Springer-Verlag, Berlin). TNT is toxic to mammals, fish, algae and other organisms and is considered a priority polluant by the United States Environmental Protection Agency (Keith and Telliard, 1979,'Priority Polluants I. A perspective view', Environ. Sci. Technol. 13, pp 416-423). Other nitroaromatic compound such as dinitrotoluenes, 2,4,6 trinitrophenol (picric acid) and pesticides/herbicides based on 2,4- dinitrophenol may also be significant pollutants.
Soil contaminated with TNT may be treated by incineration, however, this is very expensive and can give rise to undesirable products. Studies have been made investigating the transformation of TNT by micro-organisms and plants, with a view towards developing bioremediation processes (Rieger and Knackmuss,'Basic knowledge and perspectives on biodegradation of 2,4,6-trinitrotoluene and related nitroaromatic compound in contaminated soil', pp 1-18,1995, Spain, J. (ed)'Biodegradation of nitroaromatic compounds', Plenum Press N. Y.). Many organisms are capable of reducing TNT to nitroso-, hydroxylamino-and amino-derivatives. However, these compound are still toxic and are often recalcitrant to further degradation. One organism, a strain of Pseudomonas sp. designated clone A, has been shown to grow with TNT as sole nitrogen source (Duque et al, 1993,'Construction of a Pseudomonas hybrid strain that mineralises 2,4,6- trinitrotoluene', J. Bacteriol. 175, pp 2278-2283). TNT was denitrated to produce dinitrotoluenes, mononitrotoluenes and toluene. It was propose that the initial denitration step proceeds via the hydride-Meisenheimer complex of TNT (Haidour and Ramos, 1996, 'Identification of products resulting from the biological reduction of 2,4,6-trinitrotoluene, 2,4-dinitrotoluene and 2,6-dinitrotoluene by Pseudomonas sp.', Environ. Sci. Technol. 30, pp 2365-2370). This is a reduced derivative of TNT bearing a negative charge and can easily be produced by chemical reduction of TNT using boron hydrides (Kaplan and Seidle, 1970,'Studies in boron hydries. 4. Stable hydride Meisenheimer adducts', J. Org. Chem.
36, pp 937-939). Certain other bacteria have been shown to reduce TNT to the hydrie- Meisenheimer complex, but not to grow with TNT as sole nitrogen source (Vorbeck et al, 1994,'Identification of a hydride-Meisenheimer complex as a metabolite of 2,4,6- trinitrotoluene by aMycobacterium train', J. Bacteriol. 176, pp 932-934).
It is therefore an object of the present invention to provide a process for the detection and biodegradation of TNT and for the bioremediation of environments contaminated with TNT which do not suiffer from the above mentioned disadvantages.
International patent application No. PCT/GB96/01629, the contents of which is incorporated by reference herein, discloses a strain of Enterobacter cloacae, designated strain PB2, which is capable of growth with pentaerythritol tetranitrate (PETN), a nitrate ester explosive, as sole source of nitrogen for growth. An NADPH-dependent PETN reductase was purifie from this organism and was shown to liberate nitrogen as nitrite from PETN as well as from glycerol trinitrate (nitroglycerine) and other nitrate esters. The gene encoding PETN reductase, designated onr (for organic nitrate reductase) was cloned, sequenced and overexpressed in Escherichia coli. PETN reductase shows considerable promise for enzymic detection and bioremediation of nitrate esters. A culture of this organism was deposited under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the purposes of patent procedures at the UK National Collection of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen AB2 lRY, Scotland on the 14th April 1995 under deposit number NCIMB 40718. The nucleotide sequence of the onr gene showing the coding region for PETN reductase and the amino acid sequence of PETN reductase are included herewith as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.
It has now been unexpectedly found that E. cloacae PB2 is capable of growth also with the nitro-substituted aromatic TNT as sole nitrogen source. TNT is consume during growth. Dinitrotoluenes are not produced and cannot be used as sole nitrogen sources for growth, indicating that the degradation pathway followed by E. cloacae PB2 is different from that reporte for Pseudomonas sp. clone A (Duque et al, supra).
Thus according to a first aspect of the present invention there is provided an <BR> <BR> <BR> <BR> <BR> Enterobacter cloacae bacterial strain referred to as"PB2"and deposited as NCIMB40718, and mutants or variants thereof, for use in the biodegradation of TNT Cells of E. cloacae PB2 or similar organisms could be grown according to well known techniques and applied to contaminated water, soil etc. either in situ or in specialised bioreactors. Further, compositions could be produced containing E. cloacae PB2 to be added to particular environments for the biodegradation of TNT. Therefore another aspect of the present invention involves the use of E. cloacae PB2 in the preparation of a composition used for the biodegradation of TNT in an environment.
In another aspect of the invention there is provided a method for the biodegradation of TNT in an environment comprising the steps of inoculating the environment with a sample of bacterial isolate E. cloacae PB2 and allowing the isolate to degrade the TNT in the environment. A method for degradation of PETN and TNT in the same environment by the isolate is also provided. The environment could be a waste stream or could be a ground or water sample contaminated with TNT.
As a further aspect of the present invention there is provided PETN reductase <BR> <BR> <BR> <BR> having the amino acid sequence shown in SEQ ID NO: 2 or a derivative thereof for use in the biodegradation of TNT. By derivative is meant herein a version of the amino acid <BR> <BR> <BR> <BR> <BR> sequence SEQ ID NO: 2 containing insertions, deletions and/or substitutions of the amino acid sequence such that the functionality of the enzyme is retained.
This could be used for the bioremediation of a contaminated environment such as a waste stream, or a soil or water sample and could be carried out in situ or in a bioreactor.
This method of bioremediation has the aforementioned avantages i. e. that toluene and nitrotoluenes are not produced. Two further aspects of the present invention are the use of PETN reductase in preparation of a composition used for the biodegradation of TNT in an environment and a method of bioremediation of TNT in an environment comprising the steps of adding to the environment a quantity of PETN reductase enzyme of claim 4 and maintaining the mixture under conditions appropriate for degradation of the contaminant by PETN reductase enzyme. A kit for biodegradation of an environment a store of PETN reductase and a means of contacting the environment with PETN reductase in the presence of NADPH and maintaining the environment under conditions appropriate for the degradation of TNT by PETN reductase is also provided.
According to a yet further aspect of the present invention there is provided a method for the biodegradation of TNT in an environment comprising the steps of introducing to the environment a quantity of recombinant organisms expressing the onr gene having the nucleotide sequence of SEQ ID NO: 1 or a derivative thereof and maintaining the environment under conditions appropriate for degradation of the contaminant by the recombinant organism. Such organisms could include bacteria, fungi or plants and could be grown in contaminated environments such as waste streams or soil or water samples either in situ or in bioreactors. A method of biodegradation of both PETN and TNT in the same environment is also provided.
By derivative of the gene is meant herein homologues of the gene having a coding sequence which is at least 70% identical to the onr gene, involving any and all single or multiple nucleotide additions, deletions and/or substitutions thereto.
It has been shown that PETN reductase, and Escherichia coli overexpressing this enzyme, are able to reduce TNT to the hydride-Meisenheimer complex, which is further reduced to unstable, negatively-charged soluble compound. Nitrite is liberated from TNT, demonstrating removal of nitro groups. The rection products have not been identified, but similar products are formed on chemical reduction of the hydride-Meisenheimer complex of TNT using sodium borohydride. These rection products appear to be soluble and non- aromatic and to contain less nitrogen than TNT. They are therefore likely to be much less toxic and more amenable to further biodegradation than is TNT. By contrast, other enzymes active against TNT typically reduce the aromatic nitro groups to nitroso, hydroxylamino and amine groups (Rosenblatt et al, supra). The resulting nitrogen- containing aromatic compound are still highly undesirable in the environment. E. cloacae PB2, PETN reductase, and recombinant organisms expressing PETN reductase, therefore show great promise for the bioremediation of soil or water contaminated with TNT Since the initial products of reduction of TNT by PETN reductase are brightly coloured, PETN reductase may also be useful in enzyme-based assays for the presence of TNT According to a further aspect therefore, this invention concerns a method of detecting TNT in a sample comprising the steps of adding a quantity of PETN reductase, or a derivative thereof, to the sample in the presence of NADPH and detecting the occurrence of a rection. Such detection might be through detection of the oxydation of the cofactor NADPH, for example by spectrophotometric, fluorometric or luminometric methods, or through detection of the coloured products produced by enzymic transformation of the substrate, for example by visual or spectrophotometric detection of the colour produced.
Organisms overexpressing PETN reductase or the onr gene or derivatives thereof could also be used.
In a yet further aspect of the present invention there is provided a biosensor for the detection of TNT in a sample which comprises means for contacting the sample with PETN reductase enzyme or a derivative thereof in the presence of NADPH and means for detecting the occurrence of a rection, catalyse by the PETN reductase enzyme, of TNT when TNT is present in the sample.
The invention will now be described by way of reference only with reference to the accompanying drawings of which; Figure 1 shows the growth curves for growth of E. cloacae PB2 with TNT as the sole nitrogen source, Figure 2 shows the degradation of TNT during growth of E. cloacae PB2, Figure 3 shows UV-visible absorbance spectra of ion-pair HPLC peaks following reduction of TNT Figure 4 shows the development of colour and release of nitrite during reduction of TNT by PETN reductase.
Example 1-Growth of Enterobacter Cloacae PB2 with TNT as a sole nitrogen source Growth of Enterobacter cloacae PB2 with TNT as sole nitrogen source was assessed in a minimal medium with the following composition: 19.5 mM KH2P04; 30.5 MM Na2BP04; 4 ml/l trace elements (0.5 M HCI; 25 mM MgO; 20 mM CaC03; 20 mM FeSO4; 5 mM ZnSO4; 5 mM MnSO4; 1 MM CuS04; 1 MM COSSA; 1 mM H3BO4). The carbon source was 22 MM D-glucose. As an inoculum, E. cloacae PB2 was grown for 5 days at 30°C in the above medium with the addition of 15 mM NaN02 as nitrogen source. To 50 ml of medium containing no nitrogen, 0.5 mM TNT or 1.0 mM TNT as nitrogen source, 0.5 mi inoculum was added. The cultures were incubated at 30°C with rotary agitation at 150 rpm. Each day, samples of 1 ml were removed and growth was estimated by measuring light-scattering at 600 nm. Cells were then removed from the samples by centrifugation and the supernatants were stored at-20°C for HPLC analysis.
The concentration of TNT and presence of metabolites were determined by HPLC analysis using a Techsphere 50DS reverse phase column (HPLC Technology, Macclesfield, U. K.). The mobile phase consiste of 60% v/v methanol, 40% v/v water, and was delivered at a flowrate of 1.0 ml/min. Compound were detected at 260 nm. This solvent system resolved TNT, 2,6-dinitrotoluene, 2,4-dinitrotoluene, 2-nitrotoluene and 4-nitrotoluene.
For ion-pair HPLC, the same column was used, with a mobile phase consisting of 45% v/v acetonitrile, 55% v/v 20 mM tetrabutylammonium phosphate buffer, pH 7. Peaks were detected at 260 nm and 500 nm and Ut-visible spectra of peaks were measured using a Waters 994 Programmable Photodiode Array detector.
The growth curves obtained are shown as Figure 1. Growth, estimated by turbidity, was observe only in the presence of TNT and was proportional to the amount of TNT present in the growth medium. In similar experiments where TNT was replace by 2,4- dinitrotoluene, 2,6-dinitrotoluene, 2-nitrotoluene or 4-nitrotoluene, growth did not occur.
HPLC analysis showed that TNT was removed from the medium during growth.
Figure 2 shows the degradation of TNT with growth of E. cloacae PB2 with initial amounts of TNT of 0.5 mM and 1.0 mM. Peaks corresponding to dinitrotoluene and mononitrotoluene were not detected. Two peaks were detected which may represent metabolites of TNT. One of these was similar in elution position and UV-visible spectrum to products resulting from the action of cloned E. cloacae nitroreductase (Bryant et al, 1991,'Cloning, nucleotide sequence and expression of the nitroreductase gene from Enterobacter cloacae', J. Biol. Chem. 266, pp 4126-4130) on TNT. This peak may represent a stable nitroreductase product such as aminodinitrotoluene. Such products are commonly observe when bacteria are incubated with TNT (Rosenblatt et al, 1991, supra).
The second peak observe migrated at the solvent front in standard HPLC, but was retarde by the column in ion-pair HPLC in the presence of the tetrabutylammonium counter-ion, suggesting that this peak represents a negatively charged molecule. This expriment is not sufficient to determine whether or not these peaks represent products derived from TNT Example 2-Dearadation of TNT bv PETN reductase PETN reductase was purifie from recombinant E. coli bearing the plasmid pONRl by affinity chromatography (French et al, 1996,'Sequence and Properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2', J. Bacteriol. 178, pp 6623-6627).
Rection mixtures were set up containing 7 gg/ml PETN reductase, 0.2 mM NADPH and 0.05 mM TNT, 2,4-dinitrotoluene, 2,6-dinitrotoluene, 2-nitrotoluene, 4-nitrotoluene or no substrate, in 50 mM potassium phosphate buffer, pH 7, at 30°C. Oxidation of NADPH was followed based on the loss of absorbance at 340 nm. The background rate of NADPH oxidation in the absence of substrate was 0.10 ßmol NADPH. min-l. mg protein-l. This rate was not significantly enhanced in the presence of 0.05 mM 2,4-dinitrotoluene, 2,5- dinitrotoluene, 2-nitrotoluene or 4-nitrotoluene. However, in the presence of 0.05 mM TNT, the observe rate ofNADPH oxidation increased to 0.50 pmol NADPH. min-l. mg protein-', suggesting that TNT is able to oxidize the reduced form of the enzyme, presumably becoming reduced in the process. Errors in these rate measurements were estimated as less than 1%. It was further observe that rection mixtures containing PETN reductase, NADPH and TNT developed an orange colouration suggesting the formation of a coloured product from TNT. No such colouration developed in the absence of enzyme, of TNT or of NADPH.
Known reduction products of TNT include products of nitro-group reduction, such as aminodinitrotoluene, which are uncharged and essentially colourless (Rosenblatt et al, 1991, supra), and products of aromatic ring reduction, such as the hydride-Meisenheimer complet, which is negatively charged and brightly coloured (Kaplan and Seidle, 1970, supra). However, the Ut-visible absorbance spectrum of the orange product observe when TNT was reduced by PETN reductase did not match the spectrum of the hydride- Meisenheimer complex of TNT reporte in the literature.
Example 3-Nature of the products of TNT reduction.
To investigate the nature of the coloured product or products produced by the action of PETN reductase on TNT, a rection mixture was set up containing 0.02 mg/ml PETN reductase, 0.4 mM NADPH and 0.5 mM TNT in 50 mM potassium phosphate buffer, pH 7. Samples of 100 ml were taken at intervals and diluted with 1.9 ml HPLC mobile phase (45% v/v acetonitrile, 55% v/v 20 mM tetrabutylammonium phosphate buffer, pH 7). These samples were analyse by ion-pair HPLC as described in Example 1. Peaks having ultraviolet absorbance were detected at 260 nm and peaks having visible absorbance were detected at 500 nm. The UV-visible spectra of detected peaks were measured using a Waters 994 programmable photodiode array detector. A similar expriment was performed using, in place of PETN reductase, recombinant Enterobacter cloacae nitroreductase (Bryant et al, 1991, supra), a relatively well characterized enzyme which reduces the aromatic nitro groups of TNT to amino groups via nitroso and hydroxylamino intermediates.
During the reduction of TNT by PETN reductase, a UV peak at a retention time of 7.7', corresponding to TNT, decreased. Another UV peak at a retention time of 5.4' appeared and increased in size. An identical peak was observe when PETN reductase was replace by nitroreductase. This peak is presumed to represent a nitroreductase product such as hydroxylaminodinitrotoluene or aminodinitrotoluene. This suggests that PETN reductase has nitroreductase activity. In addition, with PETN reductase, six peaks with both LTV and visible absorbance were detected, with retention times of 3.0' (peak A), 3.8' (peak B), 4.2 (peak C), 4.8 (peak D), 8.6' (peak E), and 11.6 (peak F). These peaks were not observe with nitroreductase. Peak A was confounded with the peaks ofNADPH and NADP+, so that the shape of the spectrum below 400 nm could not be determined; however, the spectrum above 400 nm was identical to the spectrum of peak B in this region. The Ut-visible spectra of peaks C and D appeared to be identical to one another, as did the spectra of peaks E and F, as shown in Figure 3. It is therefore unclear whether these peaks represent six distinct compound, or three compound, each of which migrates as two peaks due to some peculiarity of the ion-pair HPLC system, such as the formation of ion-pairs with different numbers of tetrabutylammonium ions.
If rection mixtures were left for several hours, the observe peaks decreased in size, with no peaks appearing to replace them. Visible colour in the rection mixtures also faded. This suggests that the coloured products are unstable and degrade to give non- aromatic (non UV-absorbing) products.
When samples were re-analysed in the same mobile phase but with the tetrabutylammonium counter-ion omitted, all visible absorbance, presumably corresponding to peaks A, B, C, D, E and F, eluted at the solvent front. The TNT and presumed nitroreductase product peaks were unaffected. This suggests that the visible peaks A to F represent negatively charged molecules.
The UV-visible spectra of peaks E and F were distinctive and were identical to the spectrum of the hydride-Meisenheimer complex of TNT reporte in the literature (Kaplan and Seidle, 1970; Vorbeck et al, 1984, supra). As shown in Figure 3 the spectra of peaks A, B, C and D were distinctly different from this spectrum, lacking significant absorbance above 550 nm.
Comparative Example-Comparison with chemical reduction of TNT.
To determine whether peaks E and F represented the hydride-Meisenheimer complex of TNT, the authentic hydride-Meisenheimer complex was prepared by chemical reduction of TNT using sodium borohydride (Kaplan and Seidle, 1970; Haidour and Ramos, 1996, supra). To 1 ml of 10 mM TNT in acetonitrile was added 2.8 mg solid sodium borohydride (NaBH4). The rection mixture instantly developed a deep brownish- purple colour and the Ut-visible spectrum, measured in 50% v/v acetonitrile, 50% v/v water, was identical to that reporte for the hydride-Meisenheimer complex of TNT However, it was noticed that after standing at room temperature for several hours, orange colouration and a red precipitate developed in the rection mixture. If water was added to the rection mixture at an early stage, so that excess borohydride was consume through rection with water, the purple colouration was stable over days and no orange colour developed. This suggests that the orange colouration represents a slow further reduction of the hydride-Meisenheimer complex.
Chemical rection mixtures were analyse by ion-pair HPLC as described above.
Initially, TNT disappeared and peaks identical to peaks E (large) and F (small) appeared.
As the rection proceeded and orange colouration developed, peaks apparently identical in retention time and Ut-visible spectrum to peaks A, B, C and D appeared. A large ultraviolet peak lacking visible absorbance also appeared at the solvent front. No peak corresponding to the nitroreductase product peak appeared.
These results suggest that PETN reductase reduces TNT via two competing rections. In one rection, the nitro groups are reduced in a similar fashion to that seen with nitroreductase. This is not surprising since the aromatic nitro group is a facile electron acceptor and is readily reduced by a variety of enzymes (Bryant et al, 1991). In the other rection, which is predominant in the case of PETN reductase but does not occur with nitroreductase, the aromatic ring of TNT is reduced to give the hydride-Meisenheimer complex as with chemical reduction of TNT by sodium borohydride. This is further reduced to give negatively charged orange products. With PETN reductase, the reduction of the hydride-Meisenheimer complex is rapid so that the complex is seen only transiently, however, in the case of reduction with sodium borohydride, the reduction of the complex is much slower than the initial reduction of TNT so that initially the hydride-Meisenheimer complex is the only product.
Example 4-Reduction of the hydride-Meisenheimer complex of TNT bv PETN reductase.
To confirm that the orange products are produced through further reduction of the hydride-Meisenheimer complet hydride-Meisenheimer complex was prepared chemically as described above and the chemical reduction was quenched with aqueous buffer. An enzymic rection mixture was set up containing 0.4 mM NADPH, 0.04 mg/ml PETN reductase, and the amount of chemical reduction product corresponding to 2 mM TNT, in 50 mM potassium phosphate buffer, pH 7. The brown-purple colour of the chemical reduction product, presumed to be the hydride-Meisenheimer complex of TNT, was rapidly replace by an orange colour identical to that seen in enzymic reduction of TNT by PETN reductase. The IJV-visible absorbance spectrum of the rection mixture was identical to that seen during enzymic reduction of TNT. When nitroreductase replace PETN reductase, the orange colouration and the distinctive UV-visible spectrum associated with the orange products were not seen.
Example 5-Liberation of nitrite from TNT by PETN reductase It was further noted that, during enzymic reduction of TNT by PETN reductase, nitrite was liberated. A rection mixture was set up containing 0.04 mg/ml PETN reductase, 2.0 MM NADPH, and 0.5 mM TNT, in 50 mM potassium phosphate buffer, pH 7. Visible absorbance at 440 nm, corresponding to the visible absorbance peak in aqueous buffers of the orange products, was monitored. Samples were periodically removed and assayed for nitrite as follows: 6 pI was added to 594 41 water. To this diluted sample were added 200 zip of 10 mg/ml sulphanilamide in 0.68 M HCI, and 40 tl of 10 mg/ml N-(l- naphthyl) ethylenediamine in water. Visible absorbance at 540 nm was measured. Sodium nitrite was used as a standard. Results are shown as Figure 4. Over 3 h of incubation, 0.066 mM nitrite was released from 0.5 mM TNT, or 0.13 mol nitrite/mol TNT. In rection mixtures which had been left standing for several days, up to 1.0 mol nitrite/mol TNT was detected. It is not clear from these experiments whether or not this represents a limiting value.
Conversion of TNT to the hydride-Meisenheimer complex does not result in libration of nitrite. In our enzymic rection mixtures, the hydride-Meisenheimer complex appeared to be a minor and transient product. Possibly nitrite was released either during further reduction of the hydride-Meisenheimer complex to the orange products, or during breakdown of the orange products to unidentified colourless, non-W-absorbing products.
Example 6-Growth of Transgenic Plants In order to investigate the ability of transgenic plants to grow with TNT as a nitrogen source transgenic tobacco plants were produced. Tobacco was chosen due to its ease of genetic manipulation.
The onr gene was modifie by PCR to introduce a plant consensus start sequence AACAATGG which resulted in the alteration of the first amino acid from serine to alanine.
To check the activity of the modifie gene it was expressed in E. coli as described above and it was found that the activity was unaffected.
The modifie gene was introduced into tobacco (Nicotiana tabacum cv xanthi) leaf discs by Agrobacterium-mediated transformation using the binary vector method of Cleave (A. P. Cleave, 1992, Plant Mol. Biol. 20,1203-1207). Plants from 24 independent transformation events were regenerated. Genomic DNA was prepared from leaf tissue using a Phytopure kit (Scotlab). In all 24 lines the transgene was detected by PCR using the same primers as originally used for modification of the gene.
The primary plants were allowed to self fertilize and seeds were collecte. These seeds were surface sterilized and germinated in Murashige and Skoog complete medium (ICN) amended with varying concentrations of explosives. These were contraste with the growth of seeds from wild unmodified plants. TNT at 0.05 mM was found to seriously inhibit germination and growth of wild plants whereas the seeds of the transgenic plants grew comparably to seeds sown in media without explosive.
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