RU2297450C2 - Kit of lux-biosensors for heptyl detection in medium - Google Patents

Abstract

FIELD: biotechnology, gene engineering, ecology.

SUBSTANCE: Escherichia coli cells containing plasmides with luxCDABE-genes are produced by recombinant DNA method under control of PkatG, PsoxS, PrecA, PgrpE inducible stress promoters. On the base of the same kit of Escherichia coli bacterial cells is developed for heptyl detection in environment medium.

EFFECT: accelerated method for heptyl detection.

1 tbl, 4 dwg

Description

The invention relates to the assessment of the degree of environmental pollution with heptyl, and can also be used in studies of biological objects.

A known method for the determination of heptyl in the environment by chemical means [1-3]. The essence of this method lies in the photometric analysis of the decay products of heptyl or high-resolution chromatographic (gas chromatography) separation of chemical compounds with mass spectrometric detection [1], and the identification of heptyl and its decay products using NMR and infrared spectroscopy [2]. The disadvantage of chemical methods is the need to use expensive equipment, as well as the complexity of the analysis due to the high instability of heptyl [3].

Currently, biotesting methods using lux biosensors are widely used to determine environmental pollution by toxic substances. Known methods based on quenching of bioluminescence by toxicants, which use the mechanism of the inhibitory effect of toxic substances on cell metabolism, mainly on the respiratory chain, which indirectly affects the luciferase reaction, causing a decrease in the intensity of cell bioluminescence. In this series of techniques used as an express control of the toxicity of natural environments, the so-called so-called most widely used in Europe and the United States. Microtox (Microtox 5TM), which uses lyophilized marine bacteria Photobacterium phosphoreum as a biosensor. The disadvantage of this method is the non-specificity of the reaction and low sensitivity. Additional analysis is required to identify a chemical compound that caused a decrease in the intensity of cell luminescence.

Detection of toxicants using lux biosensors based on Escherichia coli bacteria containing plasmids with lux genes under the control of inducible stress promoters is also known [4]. These lux biosensors, as indicated in the cited US patent, were used to detect nitrates, phenols, gasoline, and other toxicants, but were not used to detect heptyl in the medium.

The technical result of the proposed invention is the ability to implement fast, not requiring expensive and complex equipment, both in stationary and in the field, to monitor the heptyl content in the environment.

To achieve this result, it was proposed to use a complex of lux biosensors based on Escherichia coli bacteria containing hybrid plasmids with lux genes under the control of inducible stress promoters P _katG , P _soxS , P _recA , P _grpE . In the proposed invention, a group of biosensors was used to determine heptyl, which have the property of inducing (amplifying) the signal (bioluminescence) of the cells when exposed to a toxicant, as well as high sensitivity and specificity, as it is determined by the peculiarity of the interaction of the protein receptor (repressor or activator) with a chemical compound.

The invention is illustrated by the attached graphs, where Fig. 1 shows the dependence on the time of action of heptyl (at a fixed concentration) on the biosensor P _katG :: lux; figure 2 - shows the dependence of the magnitude of the effect (with the optimal aging time of the drug) on the concentration of heptyl in the sample, also for the biosensor P _katG :: lux; figure 3 shows the reaction to heptyl (depending on the time of action) on the biosensor P _soxS :: lux; figure 4 shows the time dependence of the action of heptyl on the biosensor P _recA :: lux.

During the action of heptyl on the cell, bioluminescence is _induced in the biosensors P _katG :: lux, P _soxS :: lux, P _recA :: lux, but not P _grpE :: lux (therefore, the data for the biosensor P _grpE :: lux are not given). As can be seen from the graphs presented in figures 1-4, when holding samples with a fixed concentration of heptyl, a significant increase (about 100 times) in the intensity of bioluminescence of biosensor cells is observed over time. Figure 2 shows the dependence of the effectiveness of heptyl on its quantity in the sample for a biosensor with the promoter P _katG . Three characteristic areas can be distinguished in the curve. When the amount of heptyl in the sample is more than 20 micromoles, a significant toxic effect of heptyl on the cell is observed, which is manifested in the quenching of bioluminescence. This result is consistent with data on the lethal effect of heptyl on E. coli bacteria: bacterial death was recorded when the amount of heptyl in the sample was above 20 micromoles [5]. With heptyl amounts from about 10 to 0.1 micromole, a significant increase in bioluminescence is observed, exceeding the initial value of I _{0 by} more than 10 times. And finally, when the amount of heptyl in the sample is less than 0.01 micromole, the effect of heptyl decreases almost to the background, i.e. the threshold concentration of heptyl fixed by a lux biosensor with the P _katG promoter is 0.001 μM per sample.

In bacteria, one can distinguish regulatory systems that specifically respond to toxicants that act on: 1) cell membranes; 2) proteins; 3) a chromosome (DNA), and 4) inducing oxidative stress in the cell. It was proposed to use the P _grpE promoter as a biosensor for toxicants acting on cellular proteins. This promoter in the bacterial genome is located in front of the heat shock genes and opens only when modified, denatured proteins appear in the cell. The SOS promoter P _{recA is} used as a biosensor for DNA tropic agents. The promoter P _recA opens only upon induction of damage in the genome, i.e. in DNA molecules. For the detection of substances inducing oxidative stress in the cell (forming a hydroxyl radical in the cell, superoxide ion radical, hydrogen peroxide), the promoters P _katG and P _soxS were used. The P _katG promoter ( _OxyR activator) specifically reacts to hydrogen peroxide, organic peroxides. The P _soxS promoter opens when a superoxide-ion-radical appears in the medium.

Specific lux biosensors based on these inducible promoters are constructed in the present invention. All promoters used with the corresponding regulatory regions were obtained from the bacterial genome of Escherichia coli K12 MG1655 using the PCR method using special, synthesized primers. As a vector, a non-promoter vector was used with the ColE1 replicon and the bla gene, which determines ampicillin resistance (selective marker). The insertion of the promoter region into the plasmid was carried out at EcoRI-BamHI sites. The lux operon Photorhabdus luminescens, consisting of five genes, luxCDABE, was chosen as the lux cassette. This cassette has two advantages: firstly, luciferase Ph. luminescens (luxAB genes) is characterized by relatively high thermal stability, and, secondly, the substrate of the luciferase reaction, aliphatic aldehyde, should not be added to the cell suspension, since it is synthesized in the cell using proteins encoded by luxCDE genes [6].

The technique for measuring the effect of the toxicant on the bioluminescence of the biosensor was approximately the same for all lux biosensors.

Determination of heptyl in the medium using lux biosensors requires the following operations.

1. Preparation of samples containing appropriate biosensors;

- growing E. coli cells, which are biosensors, to an exponential phase (OD = 0.2-0.4);

- sampling of 200 μl in vials (6-8 vials for each biosensor).

2. Adding to the samples heptyl or liquid medium with heptyl in various concentrations;

- preparation of positive and negative controls for each biosensor. As a positive control, heptyl (diluted in water) at a concentration of 20 μg / ml is used (negative control is distilled water);

- adding 20 μl of the test medium to each sample of each biosensor.

(All samples should be prepared in double or triplicate and then use the average values of the obtained experimental values).

3. Measurement of bioluminescence intensities of samples for 1-2 hours for biosensors P _katG :: lux, P _soxS :: lux, P _grpE : lux. For the P _recA :: lux biosensor, the measurement should be carried out 2.5-3 hours.

4. Processing the results. The obtained graphs on control samples are compared with the test sample.

A cell culture containing a hybrid plasmid with the corresponding promoter and lux cassette was grown at 28 ° C or 37 ° C on a shaker until the early or middle exponential phase (OD = 0.2-0.4). Aliquots of this culture (200 μl each) were transferred to sterile vials and 10 μl of the test substance of the required concentration was added to them. 10 μl of distilled water was added to the control tube. Then the samples were incubated without stirring at 30 ° C and every 15 min the intensity of bioluminescence was measured at room temperature. The signal amplification was recorded after 15-20 minutes - the time required for the synthesis of luciferase. The maximum response of the biosensor, as a rule, was observed after 40-60 minutes (in the case of the P _recA promoter, the maximum signal was recorded after 90 minutes) and at the optimal concentration of the toxicant exceeded the initial signal by 100-1000 times.

As a result of our studies, it was shown that the main promoters that open when exposed to heptyl cells are the promoters P _katG , P _soxS , P _recA . Biosensor With the promoter P _grpE did not change the intensity of bioluminescence. Therefore, the data for this promoter are not shown in figures 1-4.

Table 1 shows the data on the minimum (threshold) concentrations of standard substances specific for this promoter that induce a noticeable (2–3 times) effect of enhanced bioluminescence in the designed lux biosensors (mitomycin C induces damage in DNA, ethanol in proteins , hydrogen peroxide and paraquat as a generator of superoxide-ion-radicals induce oxidative stress).

Table 1 Biosensor Standard substance Threshold concentration, M / L Bioluminescence Induction Factor P _recA :: lux Mitomycin c 10 ^-8 3.3 P _katG :: lux H ₂ O ₂ 5 × 10 ^-7 2.2 P _grpE :: lux Ethanol 0.5% 2.1 P _soxS :: lux paraquat 10 ^-7 2,3

Heptyl, apparently, unlike the compounds shown in Table 1, does not have high specificity for one or another biosensor, since due to its high reactivity (a very strong reducing agent) it can damage macromolecules. Therefore, the optimal solution to the problem of heptyl fixation in the medium is to use all the above lux biosensors.

Since the promoter P _katG opens only when exposed to hydrogen peroxide on the protein regulator OxyR (oxidation of the active center [Fe-S]), and the promoter P _soxS opens only when exposed to superoxide ion radicals, it can be considered proven that the action of heptyl on a bacterial cell is mainly determined by the formation of reactive oxygen species, in particular, hydroperoxide and superoxide-radical ion (as a result of the reduction of atmospheric oxygen to hydrogen peroxide).

Hydroperoxide, formed as a result of chemical reactions of heptyl H ₂ NN (CH ₃ ) ₂ mainly with oxygen O ₂ , opens the promoter P _katG , and is as effective as hydrogen peroxide used as a standard:

O ₂ + H ₂ NN (CH ₃ ) ₂ = O ₂ H (superoxide radical) + HNN (CH ₃ ) ₂ , and the superoxide radical quickly transforms into hydrogen peroxide:

2O ₂ H = H ₂ O ₂ + O ₂

The conversion of superoxide-ion-radical to hydrogen peroxide occurs both spontaneously and as a result of the action of the cell enzyme superoxide dismutase.

The effect of heptyl on DNA and, accordingly, on the P _recA promoter, is indirect and is determined mainly by the forming active forms of oxygen. This conclusion follows from the data on the effect of hydrogen peroxide on the biosensor P _recA :: lux, as well as from the data on the practical coincidence of the concentration dependences of the effects of heptyl and hydrogen peroxide on the biosensors P _katG :: lux and P _recA :: lux to concentrations of 10 ^-7 - 10 ^-6 M / l.

The effect of heptyl on the P _grpE :: lux biosensor is practically absent, therefore, data on the effect of heptyl on this biosensor are not given. Heptyl does not modify cellular proteins, since it does not penetrate the cytoplasm of the cell, and all is consumed (oxidized) outside the cell.

The invention allows to create a highly sensitive, cheap and fast test method based on measuring the intensity of bioluminescence.

Information sources

1. Dmitriev O.Yu., Ivanenko S.I., Ovsyannikov D.A., Smirnova S.S., Chistova Zh.A. In Sat "Proceedings of the Russian Academy of Engineering, Section" Engineering Problems of Stability and Conversion ", Issue 11." Environmental problems of the development and operation of rocket and space technology ", Moscow, SIP RIA, 2004, pp. 40-43.

2. Lopyrev V.A., Dolgushin G.V., Laskin B.M. (2001) Magazine Ros. Chem. about them D.I. Mendeleev, vol. XLV, No. 5-6, pp. 149-156.

3. Kuznetsova L.V. In Sat "Proceedings of the Russian Academy of Engineering, Section" Engineering Problems of Stability and Conversion ", Issue 12." Environmental problems of the development and operation of rocket and space technology ", Moscow, SIP RIA, 2004, pp. 24-25.

4. US patent No. 5683868, C12Q1 / 68 - prototype.

5. Anti Poso, Atte von Wright, Jukka Gynther (1995) Mutation Research. V.332. PP.63-71.

6. Zavilgelsky G. B., Zarubina A. P., Manukhov I.V. (2002) Molecular biology. vol. 36, pp. 792-804.

Claims (1)

A set of lux biosensors for detecting heptyl in a medium consisting of a sample of Escherichia coli cells transformed with a plasmid containing bacterial luxCDABE genes under the control of the P _katG promoter, a sample of Escherichia coli cells transformed with a plasmid containing bacterial luxCDABE genes under the control of a P _soxS promoter, samples of Escherichia coli cells transformed with a plasmid containing bacterial luxCDABE genes under the control of the _H promoter P; samples of Escherichia coli cells transformed with a plasmid containing bacterial luxCDABE genes under the control of the P _grpE promoter .

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