Kit of lux-biosensors for heptyl detection in medium

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

 

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

There is a method of determining heptyl environmental chemicals [1-3]. The essence of this method consists in carrying out a photometric analysis of the decay products of heptyl or high-resolution chromatographic (gas chromatography) separation of chemical compounds with mass spectrometric detection [1], and identification of heptyl and its decay products using the methods of NMR and infrared spectroscopy [2]. The disadvantage of chemical methods is the need to use expensive equipment, as well as the complexity analysis due to the high instability of heptyl [3].

Currently widespread to determine the environmental pollution by toxic substances received methods of biotesting using the lux-based biosensors. Known methods based on the quenching of bioluminescence toxicants, using the mechanism of inhibitory effect of toxic substances on cell metabolism, mainly in the respiratory chain, which indirectly affect the luciferase reaction, causing a decrease in the intensity of bioluminescence of cells. In this series of techniques used in the when asked for Express monitoring of toxicity in natural environments, most common in Europe and in the United States received the so-called "Microtox" ("Microtox 5TM), in which the biosensor is used freeze-dried marine bacterium Photobacterium phosphoreum. The disadvantage of this method is nespecificnomu response and low sensitivity. To identify chemical compounds that caused the decrease in the intensity of fluorescence of cells required additional analysis.

It is also known detection of toxicants using lux-biosensors based on bacteria Escherichia coli containing a plasmid with lux genes under the control of a stress inducible promoters [4]. These lux-biosensors as described in the above U.S. patent, were used to determine nitrates, phenols, benzene and other toxins, but has not been used for detection in the environment of heptyl.

The technical result of the proposed invention is the possibility of rapid, does not require expensive and complex equipment, both stationary and field conditions, control over the content of UDMH in the environment.

To achieve this result it is proposed to use complex lux-biosensors based on bacteria Escherichia coli containing hybrid plasmids with lux genes under the control of a stress inducible promoters PkatGPsoxSPrecAPgrpE. In the proposed izobreteny is to determine heptyl group used biosensors, having the property of inducing (gain) of the signal (bioluminescence) cells when exposed to a toxicant, as well as high sensitivity and specificity, as determined by the feature interaction protein-receptor (repressor or activator) with a chemical compound.

The invention is illustrated the accompanying diagrams, in which figure 1 shows the time dependence of the actions of heptyl (at a fixed concentration) on the biosensor PkatG::lux; figure 2 shows the dependence of the magnitude of the effect (when the optimal time-keeping of the drug) concentration of UDMH in the sample, also for biosensor PkatG::lux; figure 3 shows the response to heptyl (depending on time) on the biosensor PsoxS::lux; figure 4 shows the time dependence of the actions of heptyl on biosensor PrecA::lux.

In the process steps of heptyl on the cell occurs induction of bioluminescence in biosensors PkatG::lux, PsoxS::lux, PrecA::lux, but not PgrpE::lux (so the data for biosensor PgrpE::lux not shown). As can be seen from the graphs presented in figures 1-4, while keeping the samples with a fixed concentration of UDMH is observed over time, a significant increase (about 100 times) the intensity of bioluminescence cell - based biosensors. Figure 2 shows the dependence of the effective action which I heptyl the amount present in the sample to the biosensor with the promoter P katG. In the curve can be divided in three distinctive areas. When the number of UDMH in the sample more than 20 micromol there is a significant toxic effect of UDMH in the cell, resulting in the damping of bioluminescence. This result corresponds to the data on the lethal action of heptyl on the bacteria E. coli: the death of the bacteria was measured in number of UDMH in the sample above 20 micromol [5]. When quantities of heptyl about 10 to 0.1 micromole observed a significant increase in bioluminescence in excess of the original value of I0more than 10 times. And finally, when the number of UDMH in the sample is less than 0.01 micromole influence of heptyl reduced almost to background, that is, the threshold concentration heptyl, fixed lux-biosensor with promoter PkatG0.001 microns to test.

In bacteria, it is possible to allocate regulatory system, specifically reacting to the toxins that act on: 1) the cell membrane; 2) proteins; 3) chromosome (DNA), and (4) inducing in the cell oxidative stress. As biosensor for toxicants acting on cellular proteins, it is proposed to use the promoter PgrpE. This promoter in a bacterial genome genes located in front of "heat shock" and opens only when the cell is modified, denaturirovannykh proteins. In the role of biosensor for DNA genotype agents uses the I-SOS promoter P recA. The promoter PrecAonly opens when the induction of damage in the genome, i.e. in DNA molecules. For detection of substances inducing in the cell oxidative stress (forming in the cell hydroxyl radical, the superoxide ion-radical, hydrogen peroxide) were used promoters PkatGand PsoxS. The promoter PkatG(activator OxyR) specifically reacts to the hydrogen peroxide, organic peroxides. The promoter PsoxSopens when the environment superoxide ion-radical.

The present invention is designed based on those induced by the promoters of specific lux-biosensors. All the promoters with the relevant regulatory sites were obtained from the genome of the bacteria Escherichia coli K12 MG1655 using the PCR method with the use of special synthesized primers. As vector used besprovodnyj vector with the ColE1 replicon and bla gene determining resistance to ampicillin (selective marker). Embedding promoter region in plasmid carried out on sites EcoRI-BamHI. As lux-cassette was chosen lux-operon of Photorhabdus luminescens, consisting of five genes luxCDABE. This cassette has two advantages: first, the luciferase Ph. luminescens (luxAB genes) is relatively high thermal stability, and, secondly, to the cell suspension should not be added substrate Lucifer is Noi response - aliphatic aldehyde, as it is synthesized in the cell by using the proteins encoded by luxCDE genes [6].

Methods of measuring the impact of a toxicant on bioluminescence biosensor was approximately the same for all lux-biosensors.

The definition of UDMH in the environment using the lux-based biosensors requires the following operations.

1. Preparation of samples containing the corresponding biosensors;

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

- samples of 200 ál in vials (6-8 vial for each biosensor).

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

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

- add 20 µl sample environment for each sample of each biosensor.

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

3. Measuring intensities of bioluminescence samples within 1-2 hours for biosensors PkatG::lux, PsoxS::lux, PgrpE:lux. For biosensor PrecA::lux measurement should be 2.5-3 hours.

4. Obrabatyvatsya results. The obtained graphs on the test samples are compared with a test specimen.

Culture of cells containing the hybrid plasmid with an appropriate promoter and lux-cassette, were grown at 28°37°With the rocking until the early or mid exponential phase (OD=0.2 to 0.4). Aliquots of this culture (200 μl) was transferred into sterile vials and added them in 10 ál of test substance concentration required. In the control test tube was added 10 μl of distilled water. Then samples were incubated without agitation at 30°and every 15 min was measured intensity of bioluminescence at room temperature. The amplified signal 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 min (in the case of promoter PrecAthe maximum signal was recorded after 90 min) and with the optimal concentration of toxicant exceeded the initial signal 100-1000 times.

In our experiments it was shown that the main promoters that when exposed to cells heptyl, are the promoters of PkatGPsoxSPrecA. Biosensor With promoter PgrpEdo not modify the intensity of bioluminescence. Therefore, data on this promoter is not shown in figure 1-4.

Table 1 presents data on the minimum (threshold) to the ncentrated standard, specific promoter substances, inducing a significant (2-3 times), the amplification of bioluminescence, designed lux-biosensors (mitomycin C induces damage to DNA, ethanol - in proteins, hydrogen peroxide and paraquat as a generator of superoxide ion radicals induce oxidative stress).

Table 1
BiosensorStandard substanceThe threshold concentration, M/lFactor induction of bioluminescence
PrecA::luxMitomycin C10-83,3
PkatG::luxH2About25×10-72,2
PgrpE::luxEthanol0,5%2,1
PsoxS::luxparaquat10-72,3

Heptyl, apparently, unlike shown in table 1 compounds, does not have a high specificity to one or another of the biosensor, as well as in connection with high reaction activity (a very strong reducing agent) can damage macromolecules. Therefore, the optimal solution to the problem of fixation of UDMH in the environment is the use of all the above lux-biosensor is.

As the promoter PkatGonly opens when exposed to hydrogen peroxide for protein-regulator OxyR oxidation of the active site of [Fe-S]), a promoter PsoxSonly opens when exposed to the superoxide ion-radical, it can be considered proven that the effect of heptyl on bacterial cell is mainly determined by the formation of reactive oxygen species, in particular, gidroperekisi and superoxide ion-radical (in the recovery of oxygen to hydrogen peroxide).

Gidropress resulting from chemical reactions of heptyl H2NN(CH3)2mainly with oxygen O2opens the promoter PkatGand as effective as hydrogen peroxide, used as standard:

O2+H2NN(CH3)2=O2H (superoxide radical) +HNN(CH3)2and the superoxide radical quickly turns into hydrogen peroxide:

2O2N=N2O2+O2

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

The effect of heptyl on DNA and, consequently, the promoter PrecAthat is indirect and is mainly determined by forming reactive oxygen species. This conclusion follows from the data on the influence of peroxide water is an ode to the biosensor P recA::lux, as well as from data on the practical coincidence of the concentration dependency of the effects of heptyl and hydrogen peroxide on biosensors PkatG::lux and PrecA::lux up to concentrations of 10-7-10-6M/L.

The influence of heptyl on biosensor PgrpE::lux is practically absent, so the data on the impact of heptyl this biosensor are not given. Heptyl not modify cellular proteins, as it does not penetrate into the cytoplasm of a cell, and the whole is consumed (oxidized) outside the cell.

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

Sources of information

1. Dmitriev O.Y, Ivanenko, S., D.A. Ovsyannikov, S. Smirnov, Chistov ZH.A., " IN proc. "Proceedings of the Russian engineering Academy, Section "Engineering problems of stability and conversion", Issue 11. "Ecological problems of development and operation of rocket and space technology", Moscow, SIP RIA, 2004, p.40-43.

2. Lopyrev VA, Dolgushin, GV, luskin BM (2001) Journal of ROS. chem. of the society to them. Mendeleev, .XLV, No. 5-6, str-156.

3. Kuznetsova L.V., " IN proc. "Proceedings of the Russian engineering Academy, Section "Engineering problems of stability and conversion, Issue 12. "Ecological problems of development and operation of rocket and space technology", Moscow, SIP RIA, 2004, p.24-25.

4. U.S. patent No. 5683868, C12Q1/68 - p is totip.

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

6. Zailiyskiy G.B., Zarubina A.P., Manukhov I.V. (2002) Molecular biology. T.36, str-804.

Set lux-biosensors for the determination of UDMH in a medium consisting of samples of Escherichia coli cells, transformed with the plasmid containing the bacterial luxCDABE genes under the control of the promoter PkatGsamples of Escherichia coli cells, transformed with the plasmid containing the bacterial luxCDABE genes under the control of the promoter PsoxSsamples of Escherichia coli cells, transformed with the plasmid containing the bacterial luxCDABE genes under the control of the promoter Pgoessamples of Escherichia coli cells, transformed with the plasmid containing the bacterial luxCDABE genes under the control of the promoter PgrpE.



 

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