Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

Abstract

Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

Gianluca Tell1, Igor Paron1 and Marcello Civilini

(1)  Department of Biomedical Sciences & Technologies, Molecular Biology Section, University of Udine, Udine, Italy
(2)  Department of Food Science, Environmental Microbiology Section, University of Udine, via Marangoni 97, I-33100 Udine, Italy

Biotechnology Letters 10.1007/s10529-006-9197-1.

 

Abstract 

A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix]. PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or semisolid organic substrates biotransformation.

Keywords  Biofiltration - Nucleic acids - Organic matter - RT-PCR - Toluene/benzene-2-monooxygenase - Xylene monooxygenase

 


Introduction

The development of sensitive techniques to detect microorganisms with specific metabolisms for compounds mineralization can facilitate microorganism isolation, both in situ and in the selection phase. Optimization of the PCR (polymerase chain reaction) amplification techniques on native populations in complex solid matrices, together with their rapid execution, has allowed their application in the detection of catabolic genes to characterize the biodegrading ability of a microbial community. These techniques also allow the persistence of these properties to be determined, providing immediate indications on the effectiveness of the operative conditions when biological processes are conditioned with specific microbial inoculations.

The main objective of this study, as part of a wider experimentation on the biological treatment of volatile organic compounds (VOCs) in industrial gaseous emissions, was to set up a PCR and RT-PCR molecular method for monitoring the expression of some fundamental microbial genes involved in VOCs degradation when supported on organic solid matrices. Microbial enriched biofilter beds were used to reduce complex solvent systems containing slow-degrading aromatic compounds (xylenes often account for 20% and, together with toluene, could form 35% of total VOCs) (Civilini 2006), because it is known that acceptable removal levels cannot always be achieved for each compound and inhibitory effects on the microflora have been reported (Chang et al. 2000).


Materials and Methods

Bacterial strains and growth conditions

Several strains were isolated during biofiltration processes of mixtures and single components of VOCs contaminated air. Two strains, named 14 and C18, were selected as positive and negative controls for all molecular reactions. The strains were isolated from a mixed population of an enriched culture of samples withdrawn from bed biofilter matrices. This was generally constituted of composted organic matter (food waste, plant residues and wood chips). Enrichment cultures were set up using minimal salt basal medium (MSB) (Stanier et al. 1966) supplemented with each single solvent as carbon source and grown at 28°C. The same medium was utilized to determine microbial growth (O.D. 660 nm) and respirometric assay (Pritchard et al. 1992), while 15 g Noble agar l−1 was added to prepare plate tests with the carbon source distributed in vapor phase.

Escherichia coli TOP10 and BL21 (Stratagene, La Jolla, CA, USA) were used as a further negative control.

DNA extraction from bacterial culture

Genomic DNA of isolated strains grown in LB broth (Triptone 10 g l−1, yeast extract 5 g l−1, NaCl 10 g l−1) was extracted as described previously (Versalovic et al. 1991).

Nucleic acid (DNA and RNA) extraction and purification from organic matter samples

Extractions were performed essentially as described by Griffiths et al. (2000). Briefly, 100 mg powdered organic matrix, obtained from each bioreactor after freezing, lyophilization and further homogenization, were dissolved in 0.1 ml CTAB extraction buffer and 0.1 ml of phenol/chloroform/isoamyl alcohol (25:24:1 by vol.) (pH 8.0).

After extraction-purification steps, pellets containing nucleic acids were washed with 70% (v/v) ethanol, air dried and resuspended in 30 μl Tris/EDTA buffer (pH 7.4 and RNase free).

The extracted DNA and RNA quality was further verified by analysis on agarose gel stained with ethidium bromide (0.5 μg/ml).

Preparation of purified RNA for RT-PCR analysis

Extracted nucleic acids, 30 μl, were treated with (3 U) DNase (Sigma and RNase-free) for 1 h at 37°C. The reaction mixture was then mixed with guanidine thiocianate (GTC) up to 200 μl and 200 μl sodium acetate, 2 M pH 4.0, followed by 40 μl chloroform/isoamyl alcohol (24:1) and 200 μl phenol acid. The suspension was then vigorously stirred and placed on ice for 15 min. Subsequently, it was centrifuged at 13,00 wg for 10 min (4°C). To the collected supernatant, 1 vol. ethanol was added and then frozen for 30 min at −20°C, followed by centrifugation at 13,000 wg for 20 min (4°C). The pellet of RNA was further washed in 70% (v/v) ethanol and again centrifuged at 13,000 g for 5 min (4°C). At the end, the pellet was resuspended in 30 μl diethylpyrocarbonate (DEPC) treated water.

The RNA presence and quality was estimated on agarose gel (0.5–1% w/v) in 15% (v/v) formaldehyde, ethidium bromide, 0.5 μg/ml, run in MOPS buffer (20 mM, pH 7.0, 10 mM sodium acetate; 1 mM EDTA, pH 8.0).

Primers and PCR amplification of 16S rDNA and catabolic enzymes from genomic DNA

The 16S rDNAs were selectively amplified (Gene Amp PCR System 2400, Perkin Elmer thermal cycler) from purified genomic DNA or from boiled cells routinely cultured on LB broth. Oligonucleotide primers were designed to anneal to conserved positions in the 3′ and 5′ regions of bacterial 16S rRNA genes (Al-Robaiy et al. 2001). The forward primer prbfo corresponding to position 509–525 of E. coli 16S rRNA whose upper sequence was 5′-ACTACGTGCCAGCAGCC-3′. The reverse primer was prbre 5′-GGACTACCAGGGTATCTAATCC-3′ corresponding to the complement of position 784–805 of E. coli 16S rRNA. The amplification product thus resulted as 297 bp.

The reaction mixture, in a final volume of 100 μl, was placed in 0.2 ml thin wall Eppendorf test-tubes. The reaction mixture, contained Taq Polimerase (Perkin-Elmer) 2.5 U, template DNA solution (20–200 ng genomic DNA), 0.5 μM of each primer, 4% (v/v) DMSO, MgCl2 1.5 μM and 10 μl buffer 10× (Perkin-Elmer). Amplification conditions were as follows: a preliminary denaturation of 10 min at 94°C followed by 40 cycles of 45 s at 94°C (denaturation), 30 sec at 66°C (annealing), 30 s at 72°C (extension) and a final cycle of 8 min at 72°C. PCR products were electrophoresed at 10 V cm−1 in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).

The primers to amplify catabolic enzyme genes have been chosen to identify the key enzymes involved on the xylene and toluene aerobic degradation pathways. Due to the presence of various isoforms of the key enzymes in different bacterial species, the choice of primers has been limited to extremely conserved DNA regions using the BLAST program analysis of the National Center for Biotechnology Information of the National Institutes of Health (http://www.ncbi.nlm.nih.gov/blast).

For xylene pathways, the sequence of PCR primer, xylfwd, was 5′-CATTCTTTCTTTGGCTTGGCTTAGTGG-3′, corresponding to nucleotide 4988–5014 of xylene monooxygenase hydroxylase component (xylM) of AF019635 sequence, and the primer, xylrev, was 5′-CCTCAATCTTTATCGCATCTTTGACGG-3′, corresponding to nucleotide 5256–5230 of the same sequence. For the toluene degradation pathway, the alpha subunit-terminal oxygenase component, tbmD gene, of toluene/benzene-2-monooxygenase (Johnson and Olsen 1995) was chosen to design the following primers: tol2fwd primer sequence 5′-CGCTGGACTGACAAGTGGTTCTGG-3′, corresponding to 2838–2861 of L40033 Gene Bank sequence, and tol2rev reverse primer 5′-TTCTCCGAGAGCCATTGCATCTCTT-3′, corresponding to 3148–3124 position of the same sequence.

The reaction mixture components were the same as those described above, with amplification conditions as follows: a preliminary denaturation of 120 s at 94°C followed by 35 cycles of 45 s at 94°C, 50 s at 58°C, 60 s at 72°C and 8 min at 72°C (extension). PCR products were electrophoresed at 10 V cm−1 in 1.3% agarose in TAE buffer containing ethidium bromide (0.5 μg/ml).

RT-PCR procedure

The RT-PCR were performed on the extracted RNA, after a preliminary phase of denaturation at 70°C for 5 min followed by rapid cooling on ice. The reverse-transcription reactions were performed in 0.2 ml thin wall Eppendorf tubes in 50 μl final volume, using a mixture of exaprimers (Invitrogen, Milan, Italy) at 10 ng/μl, 200 μM of every dNTP, 200 U of M-MLV-RT (Invitrogen, Milan, Italy), 4 mM DTT, 40 U RNase inhibitor (Invitrogen, Milan, Italy), and 10 μl 5× buffer (Invitrogen, Milan, Italy). The reverse-transcription reaction was performed on 5 μl of the extracted RNA at 37°C for 1 h. The PCR was performed on 5 μl of produced cDNA as described above. Reaction tests where the sample had been omitted and replaced with an equal volume of water were used as negative control, and the presence of the rRNA 16S bacterial gene was tested as positive control.


Results and Discussion

Bacterial strains

The application of sensitive methods requires efficient negative and positive controls to determine both the reaction efficiency and the cross contaminations in all analytical procedures.

The ability to degrade and/or utilize methylbenzenes such as toluene, ortho-, meta-, para-xylene, pseudocumene (1,2,4-trimethylbenzene), hemellitol (1,2,3-trimethylbenzene), mesitylene (1,3,5-trimethylbenzene) or 1,2,3,4-tetramethylbenzene is exhibited by a wide variety of general bacteria (Table 1) (Lang 1996, Fredrickson et al. 1995). Many strains showed multiple sources of carbon requirement but only two bacteria, strains F199 (Fredrickson et al. 1991) and a mutant of strain OXI (Di Lecce et al. 1997), utilized toluene and all isomers of xylene as a sole carbon and energy source.

Several different pathways have been proposed in a variety of different microorganisms and, during the past three decades, much research has been devoted to elucidating the aromatic hydrocarbons metabolisms (Smith 1990) and genetic relationship (Singer and Finnerty 1984). Based on studies of these properties, interesting conclusions were drawn by Van der Meer et al. (1992). Regarding mechanisms of genetic adaptation, the assumptions that “common ancestral genes have spread among different microorganisms”, “the divergence of descendants does not necessarily correspond to the evolutionary distances determined from those organisms” and “the organization of catabolic gene suggest that several different gene clusters may be combined as modules, to which other peripheral genes may be added producing expansion of the existing degradative pathways” have been particularly illuminating.

Considering the convergent metabolic pathways, our purpose was to evaluate specific activities of single compounds within the mono aromatic degradation pathway. The specificity of the inductive mechanism was evinced by the use of similar molecules such as xylenes and toluene. For this purpose, we chose two strains with the characteristics described in Table 2. Microbial growth in the presence of VOCs as sole carbon source was evaluated by optical density and confirmed with respirometric assay. Because of the high VOC volatility, it was not possible to determine the exact C/CO2 conversion rate with the latter method (data not shown).

Strain 14 was used as positive control for xylene enriched matrices and strain C18 was used as a positive control for growing on toluene enriched matrices. In addition, two E. coli strains (BL21 and TOP10), grown in the same matrices, were used as xylene and toluene gene negative controls (see below).

Design and test of primers for amplification of specific degradative enzymes of xylene and toluene pathways

In order to test the expression of genes coding for specific degradative enzymes of xylene and toluene pathways, different primers were synthesized to amplify conserved portions of xylene monooxygenase hydrolase component (xylM, AF019635) and the alpha subunit-terminal oxygenase component of toluene 2 monoxygenase (tmbD, L40033).

Two pairs of primers (see Table 3 for details) resulted as highly specific for the chosen genes. In fact, by using genomic DNA from C18 (Tol+) and 14 (Xyl+) isolated strains as template, these primers resulted in specific amplifications of xylM and tmbD genes by PCR. In the case of the bacterial DNA of the strain isolated for growing on toluene (C18), PCR amplification gave rise to a specific amplified product corresponding to 311 bp of toluene 2 monooxygenase partial gene (primers tol2fwd and tol2rev). In the case of bacterial DNA deriving from the strain isolated for growing on xylene (strain 14), PCR amplification with primers xylfwd and xylrev gave a specific amplification product corresponding to 269 bp of xylene monooxygenase partial gene (not shown). The specificity of the designed primers was demonstrated by performing PCR reactions with the two pairs of primers with DNA extracted from E. coli cells (strain BL21 and TOP10), which gave no amplification products. The correctness of the amplification products was confirmed by DNA sequencing of each one.

PCR amplification analysis on nucleic acid (DNA and RNA) extracted from solid biofilter matrices

Several different organic samples were withdrawn from a laboratory scale biofilter fed with single VOCs for several months. A methodology was set up to co-extract and purify the nucleic acids from these solid matrices to avoid contamination from organic and composed aromatic substances (data not shown). The amount of DNA extracted, as judged from serial dilutions of the samples and comparative electrophoresis gel analysis with a known amount of same size DNA and RNA standards, was about 100–500 ng DNA (10 kb) and 0.5–2 μg rRNA 16S from 100 mg matrix (data not shown).

To evaluate the extraction method efficiency and monitor the expression of xylM and tmbD genes, nucleic acids extracts originated from xylenes, methyl ethyl ketone, butyl acetate, ethyl acetate adapted matrices, were split into two aliquots to prepare pure template of DNA or RNA. In order to obtain pure DNA, half the sample was incubated at 37°C with RNAase (Sigma) at 100 μg/ml for 10 min. After further extraction with phenol/chloroform, the sample was used for the PCR reactions.

The presence of bacterial strains selected for their ability to form degradative pathways of xylene and toluene was then assayed by PCR on extracted DNA. As shown in Fig. 1, panels (a) and (b), efficient amplification of the 16S rDNA partial gene, for all the extracted samples harvested from the various matrices, confirmed the standardization of the extraction and purification methods. The possible presence of the two isolated bacterial strains was confirmed by PCR amplification of xylene monooxygenase (Panel a) and toluene 2 monoxygenase (Panel b) partial genes. PCR analysis confirmed the presence of xylene monooxygenase gene only in nucleic acids purified from the xylene pre-adapted matrix (Lane 1), while it was absent in the other samples. This evidence confirmed the high VOC selective pressure operated on the microorganisms subsequently grown on several matrices. Evaluation of the possible presence of the gene for toluene 2 monoxygenase was used as a control for method specificity because toluene is an aromatic compound similar to xylenes. The complete absence of this gene, in the nucleic acids purified material from xylene pre-adapted matrices (Panel b), resulted as a useful negative control regarding process selection within the matrix.

Reverse transcription and amplification of a specific fragment of cDNA starting from previously purified mRNA.

In order to estimate the expression of the xylene monooxygenase gene at mRNA level, the RNA purified from the xylene adapted matrix was converted to cDNA and used as a template for the PCR reactions. The RNAs for reverse-transcription were obtained by treating one out of two aliquots of all samples, extracted and purified from different matrices, with 5 U DNase RNase-free (Sigma, Milan, Italy), at 37°C for 30 min. Subsequently, the reverse-transcription reactions and PCR on the obtained cDNA were performed as described in Materials and methods.

Figure 2 shows the results of RT-PCR reactions. Efficient amplification of xylene monooxygenase was obtained only from samples originating from xylene adapted matrix (panel a, lane 1), confirming the sensitivity and specificity of the procedure to evaluate the expression of this gene. On the contrary, toluene 2 monooxygenase expression was, as expected, not revealed (data not shown). In any case, as a control of the RT-PCR reaction, the expression of rRNA 16S was always evaluated to confirm the integrity of the material and microorganisms viability. In each analysis, particular attention was paid to ensuring that there was no DNA cross-contamination in the RNA sample, by carrying out the RT-PCR reactions on RNA samples in the absence of the reverse transcriptase enzyme (Fig. 2, panel b). The lack of the transcript for thmD gene, as demonstrated by the absence of amplification product, might therefore suggest the absence of this enzyme. Further analysis at protein level will address this issue.

The main difficulty in applying nucleic acids technology to microbial traceability in environmental solid matrices is due to the fact that the common protocols for nucleic acids extraction in denaturing conditions involve a significant contamination of the samples with organic and composed aromatic substances released by organic matter. These compounds may significantly alter further PCR analysis by inhibiting the reverse transcriptase activity and DNA polymerase, thus affecting performance. Moreover, the standard procedures have been shown to be not particularly efficient in the extraction and purification of good quality nucleic acids, possibly because of the co-extraction of active DNAse and RNAse present in the matrices (Griffiths et al. 2000). 

 


Conclusions

Conclusions

Based essentially on sample pre-treatment procedures, this paper describes a protocol for co-extraction and PCR analysis of purified DNA/RNA from complex organic matter matrices. It could be of great practical interest in biofiltration technology, in which the high aromatic concentration in exhaust gas reduces biological process yields. Management of biological processes is frequently controlled just by monitoring physical–chemical parameters, with biological parameters only being estimated as later confirmation. An improvement of the qualitative and quantitative procedures for monitoring microbial activities could thus have direct real time applications to help in the understanding of the biodynamic requirements and maintain the right growth conditions to maximize the elimination capacity of each polluting compound. This is particularly important to track the effectiveness of specific inoculation in a heterogeneous microbial community.

For these reasons, this work, together with other biotechnological strategies, such as the use of monoclonal antibodies raised against catabolic enzymes (Civilini et al. 2000), is suitable for studying microbial community activities and of help in setting up a standardized procedure for bioremediation.

 

Acknowledgements  
 
This work was supported by Friuli Venezia Giulia Council (L.R. FVG 26.2.2001 n.4, Art. 7, c.45 and L.R. FVG 3/98, Art.16 – 2002) and CRABO group grants.

References

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Civilini M (2006) Multiple microbial activities for VOCs reduction by biofiltration. J Air Waste Manage Assoc 56:922–930
 
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Table 1

List of some microorganisms degrading alkylated benzenes

Azoarcus denitrificans

Bacillus benzoevorans

Burkholderia cepacia

Burkholderia sp.

Delftia acidovorans

Desulfobacula toluolica

Nocardia sp.

Pseudomonas fluorescens

Pseudomonas mendocina

Pseudomonas putida

Pseudomonas sp.

Ralstonia basilensis

Ralstonia eutropha

Ralstonia pickettii

Rhodococcus rhodochrous

Rhodococcus ruber

Rhodococcus sp.

Rhodococcus zopfii

Sphingomonas aromaticivorans

Sphingomonas paucimobilis

Sphingomonas stygia

Sphingomonas yanoikuyae

Thauera aromatica

Xanthobacter autotrophicus


Table 2

Characteristics and growth pattern of the isolated strains on different carbon source

 

Strain

Colonya

Cell

Carbon sourcea b

morphology

colour

morphology

Gram

TOL

EPA

XYL

C18

irregular

white

Irregular rods

+

+

14

irregular

white

rods

+

+

+


Table 3

Characteristics of the primers used in this study

 

Gene

Gene name, accession code

Primers Sequences

Position on gene

Length amplificate (bp)

Xylene monooxygenase hydroxylase component

xylM, AF019635

Fw: 5′-CATTCTTTCTTTGGCTTGGCTTAGTGG-3′

4,988–5,014

269

Re: 5′-CCTCAATCTTTATCGCATCTTTGACGG-3′

5,256–5,230

α subunit of toluene/benzene-2-monooxygenase

tbmD, L40033

Fw: 5′-CGCTGGACTGACAAGTGGTTCTGG-3′

2,838–2,861

311

Re: 5′-TTCTCCGAGAGCCATTGCATCTCTT-3′

3,148–3,124


Figures

mcith_9197_Fig1.gif Figure 1 1.5% agarose gel showing PCR amplifications with 16S rDNA positive control primers and differential target primers. Panel (A): amplifications with prbfo–prbre (16S-rDNA) and xylfwd–xylrev (xylM) of extracted DNA from xylene Organic matter (OM) (lane 1), methyl ethyl ketone OM (lane 2), butyl acetate OM (lane 3), ethyl acetate OM (lane 4). Panel (B): image of the 16S rDNA and tol2fwd - tol2rev primers amplicons from total DNA extracted from the C18 strain as a positive control (lane 1), xylene OM (lane 2), methyl ethyl ketone OM (lane 3), butyl acetate OM (lane 4), ethyl acetate OM (lane 5)

(Click image to enlarge)

mcith_9197_Fig2.gif Figure 2 1.5% agarose gel showing RT-PCR amplifications with 16S rDNA positive control primers (prbfo–prbre) and xylfwd–xylrev (xylM) target primers starting from purified RNA. Panel (A): RT-PCR of extracted and purified RNA from xylene organic matter (OM) (lane 1), methyl ethyl ketone OM (lane 2), ethyl acetate OM (lane 3). Panel (B): RT-PCR reaction controls of the previous experiment. Image of the 16S rDNA and xylM PCR amplifications where reverse transcriptase enzymes were omitted and starting from purified RNA extracted from xylene O.M. (lane 2), methyl ethyl ketone OM (lane 3), ethyl acetate OM (lane 4); previous PCR product where template was omitted is shown in lane 1. In all samples the primers utilized to amplify the two genes are visible

(Click image to enlarge)

 


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