CN105803039A - Application of gene engineering bacteria in detecting total toxicity of water - Google Patents

Application of gene engineering bacteria in detecting total toxicity of water Download PDF

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CN105803039A
CN105803039A CN201610236068.9A CN201610236068A CN105803039A CN 105803039 A CN105803039 A CN 105803039A CN 201610236068 A CN201610236068 A CN 201610236068A CN 105803039 A CN105803039 A CN 105803039A
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余登斌
雍阳春
翟俊峰
白露
刘玲
董绍俊
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Changchun Institute of Applied Chemistry of CAS
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Abstract

The invention discloses application of gene engineering bacteria in detecting the total toxicity of water, and belongs to the technical field of water detection. The problem that in the prior art, a microbiological water toxicity detection method based on gene engineering is high in cost as expensive optical spectrum instruments and substrates are used is solved. According to the application of gene engineering bacteria in detecting the total toxicity of the water, firstly, the gene engineering bacteria capable of secreting electronic media are inoculated into a culture medium capable of guaranteeing normal reproduction of the gene engineering bacteria; then, the culture medium is sub-packaged into two or more samples in an isovolumetric mode, deionized water with the volume of V is added into the control sample, a to-be-detected water sample with the volume of V is added into the detection sample, and the gene engineering bacteria are cultured; finally, the color of the control sample and the color of the detection sample are observed, it is judged that the to-be-detected water sample has no toxicity if the color of the detection sample and the color of the control sample are the same, and it is judged that the to-be-detected water sample has toxicity if the color of the detection sample and the color of the control sample are different. It is unnecessary to use expensive substrates or instruments in the application process, and the result can be directly observed with the naked eyes.

Description

Genetically engineered bacteria application in the total toxicity detection of water body
Technical field
The invention belongs to water body detection technique field, be specifically related to the application in the total toxicity detection of water body of a kind of genetically engineered bacteria.
Background technology
Fast development along with modern chemical industry agricultural, while human living standard obtains improving, environment is also affected by increasingly severe pollution, especially water environment, substantial amounts of Organic substance, heavy metal, pesticide etc. enter in water environment, become the main source of water pollution, and by the safe diet of amplification and the accumulation serious threat people, animal layer by layer of food chain.Therefore, the water in order to tackle sternness pollutes challenge, and in recent years, the total toxicity detection of various water bodys, monitoring technology develop rapidly, become whether people's early warning water environment is polluted, and judge the important means of contaminated degree.
Water body toxicity detection method of the prior art, the main rationalization method of mensuration of water body toxicity and biological method.Chemical Physics analyzes the content of main component in method energy quantitative analysis pollutant, but can not directly and complete and ground reflects the various noxious substances combined influence to environment.And harmful chemicals pollutant and organic pollutant category are various in water, most of Organic substances content in water is atomic again, will all analyze detection and study these pollutant components, and workload is very huge.Biological detection can the comprehensive function of all components in direct reaction Complex water body environment, it is possible to making up the deficiency of Physico-chemical tests method, therefore in water pollution research, it has become as one of detection and the important means evaluating water body environment.
Prior art, the method for biological detection water body toxicity mainly has photobacteria detection, algae detection, Fish detection, the detection of water flea class, microorganism detection etc..Wherein, photobacteria detection easy and simple to handle, quick, sensitive, expense is low, practical, but if it is desired to ensure reliable quality control, maintenance period is long.Algae detection individuality is little, breeding is fast, toxicity is sensitive, it is easy to separation and Culture, but workload is big, and test period is long.Fish detection water environment is very sensitive, is widely used, and however it is necessary that cultivation Fish, and experimental period is long.The detection of water flea class is bred soon, is easily cultivated, and to multiple poisonous substance sensitivity, easily observes, but result is had impact by the age of flea class and classification, it is difficult to judge whether flea class is in suitable state, and complete these and judge whether to long-term experience accumulation.It is fast that microorganism detection has detection speed, good stability, and automaticity is high, and selectivity is high, and highly sensitive, cost is low, and testing result is ratio equity advantage easily, and the advantage combining several detection method in recent years, gets the attention.Wherein, it is a new study hotspot based on genetically engineered microorganism water body toxicity detection method.As introduced in microbial body, there is promoter (Promotor) and the plasmid of reporter gene (Reporter), promoter is fabA such as, dnak or grpE etc., reporter gene is mainly lacZ (beta galactosidase) or gfp (having the luciferase of photoluminescent property) etc.；When toxic material exists, toxicity molecule can be responded by promoter, thus inducing the functional enzyme that synthesis reporter gene is corresponding, make microorganism can synthesize corresponding enzyme, and Substrate hydrolysis become the material that can electrochemically detect or sends fluorescence signal (Tessema, D.A., etal., Freeze-dryingofsol gelencapsulatedrecombinantbioluminescentE.colibyusinglyo-protectants.SensorsandActuatorsB:Chemical, 2006.113 (2): p.768-773.).This detection method can react whether water body has toxicity, but this detection process needs to use fluorescent instrument and expensive substrate, and testing cost is higher, is therefore unsuitable for extensive use.
Summary of the invention
Present invention aim to address and prior art uses based on the water body toxicity detection method of genetically engineered microorganism expensive spectral instrument and substrate, the technical problem that cost is high, it is provided that the application in the total toxicity detection of water body of a kind of genetically engineered bacteria.
The present invention provides the application in the total toxicity detection of water body of a kind of genetically engineered bacteria, and step is as follows:
Step one, genetically engineered bacteria is inoculated into ensure that in the culture medium that this genetically engineered bacteria is normally bred, obtain postvaccinal culture medium；
Described genetically engineered bacteria can autocrine electron mediator；
Step 2, postvaccinal culture medium equal-volume being packed as two parts of samples, it is the deionized water of V that a sample adds volume, obtains control sample, and another part of sample adds the water sample to be measured that volume is V, obtains detection sample；
Or postvaccinal culture medium equal-volume is packed as the sample of more than three parts, and it is the deionized water of V that a sample adds volume, obtains control sample, remaining sample is separately added into the water sample to be measured of same concentrations that volume is V or variable concentrations, obtains detection sample；
Under step 3, same culture conditions, cultivate control sample and detection sample, observe control sample and the color of detection sample, if the detection sample after cultivating and control sample solid colour, then water sample to be measured is nontoxic, if the detection sample after cultivating is inconsistent with control sample color, then water sample to be measured is poisonous.
Preferably, described genetically engineered bacteria is obtained by rhlIR gene cluster process LAN in pseudomonas aeruginosa P.Aeruginosa.
Preferably, in described step one, ensure the mixture of the antibiotic that culture medium is sterilizing that this genetically engineered bacteria normally breeds and the culture medium of sterilizing, described antibiotic is tetracycline, after mixing with the culture medium of sterilizing, and final concentration of 100-200ug/mL, or it is tetracycline and ampicillin mixture, after mixing with the culture medium of sterilizing, the final concentration of 100-200ug/mL of tetracycline, the final concentration of 50-150ug/mL of ampicillin.
Preferably, described culture medium is the one in PB culture medium, M9 culture medium, LB culture medium, PYO culture medium.
Preferably, the culture medium of described sterilizing is that at 121 DEG C, under 1 atmospheric pressure after sterilizing 20min, cooling obtains by by ultrasonic for culture medium mixing, sealing.
Preferably, the antibiotic of described sterilizing is by being dissolved in deionized water by antibiotic, then obtains with the membrane filtration of 0.05～0.5 μm.
Preferably, the condition of culture of described step 3 is: with the rotating speed of 100～250rpm, control sample and detection sample are cultivated 10～35h in the constant-temperature table of 10～45 DEG C.
Preferably, described incubation time is 15h, and cultivation temperature is 30 DEG C.
Compared with prior art, the invention has the beneficial effects as follows:
The application in the total toxicity detection of water body of the genetically engineered bacteria of the present invention, use self can secrete the genetically engineered bacteria microorganism subject as the total toxicity detection of water body of electron mediator, in the total toxicity detection of water body, signaling molecule with the electron mediator of bacterial secretory for the total toxicity detection of water body, detection process need not use expensive substrate, and electron mediator has the feature of significant color, can with the naked eye direct observed result, reduce the use cost of the total detection method of toxicity of the water body based on genetically engineered microorganism significantly, there is obvious cost advantage.
Accompanying drawing explanation
Fig. 1 is the flow chart of the genetically engineered bacteria of present invention application in the total toxicity detection of water body；
Fig. 2 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 1；
Fig. 3 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 2；
Fig. 4 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 3；
Fig. 5 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 4；
Fig. 6 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 5；
Fig. 7 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 6；
Fig. 8 is the testing result of the genetically engineered bacteria detection total toxicity of water body of embodiment 7.
Detailed description of the invention
In order to further appreciate that the present invention, below in conjunction with detailed description of the invention, the preferred embodiments of the invention are described, but it is to be understood that these describe simply as further illustrating the features and advantages of the present invention, rather than the restriction to patent requirements of the present invention.
The application in the total toxicity detection of water body of the genetically engineered bacteria of the present invention, genetically engineered bacteria can autocrine electron mediator, when water body contains toxicant, breeding and the activity of antibacterial are affected, and the electronic media bulk concentration of secretion tails off, and electron mediator has the feature of significant color, can with the naked eye direct observed result, therefore the detection sample having color distortion with control sample is poisonous water body, and color distortion is more big, and toxicity is more big.
Technical scheme is as it is shown in figure 1, be first inoculated into genetically engineered bacteria and ensure that in the culture medium that this genetically engineered bacteria is normally bred.Then postvaccinal culture medium equal-volume is packed as the sample of more than two parts.If culture medium equal-volume is packed as two parts of samples, then a sample adds volume is the deionized water of V, obtains control sample, and another part of sample adds water sample to be measured, obtains detection sample；If culture medium equal-volume is packed as the sample more than two parts, it is the deionized water of V that a sample adds volume, obtaining control sample, remaining sample is separately added into the water sample to be measured that volume is V and (can be the water sample to be measured being separately added into same concentrations, improve detection accuracy；The water sample to be measured of variable concentrations can also be separately added into, carry out gradient detection, according to concentration change, search out the Monitoring lower-cut to institute's water sampling, it is also possible to the corresponding relation according to concentration and color, obtain standard color comparison card), obtain detection sample.Finally, under same culture conditions, cultivate control sample and detection sample, when water sample to be measured is toxic samples, toxicant can suppress breeding and the activity of antibacterial, thus causing that the amount of the electron mediator of bacterial secretory reduces, observe control sample and the color of detection sample, it is possible to the total toxicity of water body is detected, if the detection sample after cultivating and control sample solid colour, then water sample to be measured is nontoxic, if the detection sample after cultivating is inconsistent with control sample color, then water sample to be measured is poisonous.It should be noted that in said process, water sample to be measured is unique influence factor.
As long as in the present invention, engineered bacterial can be secreted electron mediator and can be achieved with the present invention and detect the effect of the total toxicity of water body, it does not have particular restriction.nullBut study through inventor，Following genetically engineered bacteria can be good at realizing the effect above，Therefore，Genetically engineered bacteria of the present invention obtains preferably by rhlIR gene cluster process LAN in pseudomonas aeruginosa P.Aeruginosa (preserving number CGMCC1.860)，Concrete transformation process is referred to document: a kind of quantization (Ageneticallyengineeredwhole-cellpigment-basedbacterialbi osensingsystemforquantificationofN-butyrylhomoserinelact onequorumsensingsignal based on genetically engineered cell chromobacterium biological sensing system to butyryl homoserine lactone colony induction signaling，BiosensorsandBioelectronics25(2009)41-47).
As preferably, the present invention provides the application in the total toxicity detection of water body of a kind of said gene engineered bacterial, and step is as follows:
Step one, by ultrasonic for culture medium mixing, sealing, at 121 DEG C, sterilizing 20min under 1 atmospheric pressure, obtain the culture medium after sterilizing, then cool down standby；
Wherein, culture medium is not particularly limited, employing can be cultivated the culture medium of corresponding gene engineered bacterial and all may be used, it is preferred to the one in PB culture medium, M9 culture medium, LB culture medium, PYO culture medium, above-mentioned several culture medium all can be obtained by those skilled in the art's well-known way；
Step 2, being dissolved in deionized water by antibiotic, obtain antibiotic solution, then with the membrane filtration of 0.05～0.5 μm, obtain the antibiotic after sterilizing, it is standby to be placed in 4 DEG C of refrigerators cold preservation；
Wherein, antibiotic is tetracycline or tetracycline and ampicillin mixture；
Step 3, in the super-clean bench of sterilizing, antibiotic after sterilizing is mixed with the culture medium after sterilizing, obtain mixing wild Oryza species, when antibiotic is tetracycline, in mixing wild Oryza species, the concentration of tetracycline is 100-200ug/mL, when antibiotic and ampicillin mixture, in mixing wild Oryza species, the concentration of tetracycline is 100-200ug/mL, and the concentration of ampicillin is 50-150ug/mL；
Step 4, in the super-clean bench of sterilizing, genetically engineered bacteria is inoculated in the mixing wild Oryza species of step 3, after mixing, culture medium equal-volume is packed as two parts of samples, it is the deionized water of V that a sample adds volume, obtain control sample, another part of sample adds the water sample to be measured that volume is V, obtain detection sample, or culture medium equal-volume is packed as the sample more than two parts, it is the deionized water of V that a sample adds volume, obtain control sample, remaining sample is separately added into the water sample to be measured that volume is V, obtain detection sample, then control sample and detection sample are cultivated 10～35h with the rotating speed of 100～250rpm in the constant-temperature table of 10～45 DEG C, preferred incubation time is 15h, cultivation temperature is 30 DEG C.
The color of step 5, observation control sample and detection sample, if the detection sample after cultivating and control sample solid colour, then water sample to be measured is nontoxic, if detection sample and control sample color after cultivating are inconsistent, then water sample to be measured is poisonous.If it is desired to obtain more accurately as a result, it is possible to use spectral instrument or electrochemical apparatus.
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
Genetically engineered bacteria application in the total toxicity detection of water body:
Step one, take the PB culture medium of 1L, seal with ventilated membrane, at 121 DEG C, sterilizing 20min under 1 atmospheric pressure, cool down standby；
Step 2, preparation 50mL antibiotic, ultrasonic mixing, and degerming with 0.22 μm of membrane filtration, it is standby to be placed in 4 DEG C of refrigerators cold preservation, and antibiotic is the mixture of tetracycline and ampicillin, wherein, the concentration of ampicillin is 10mg/mL, and the concentration of tetracycline is 15mg/mL；
Step 3, in the super-clean bench of sterilizing, 0.9mL antibiotic after degerming is added in the culture medium after 89.1mL sterilizing；
nullStep 4、In the super-clean bench of sterilizing，Genetically engineered bacteria (is obtained by rhlIR gene cluster process LAN in pseudomonas P.aeruginosaCGMCC1.860，The electron mediator of the phenazine structure of high concentration can be secreted) it is inoculated into step 3 and contains in antibiotic culture medium，After mixing，Culture medium equal-volume is packed as five parts (every part of 18mL)，It is the deionized water of 2mL that a copy of it adds volume，All the other four parts add volume is 2mL，Concentration is 25mg/L respectively，50mg/L，100mg/L，The 3 of 200mg/L,5-chlorophenesic acid (3,5-DCP)，Namely in five parts of samples 3,The concentration of 5-DCP respectively (a) 0mg/L,(b)2.5mg/L,(c)5mg/L,The 3 of (d) 10mg/L and (e) 20mg/L,5-DCP，30 DEG C of constant-temperature tables cultivate 15h with 220rpm rotating speed.
Step 5, with the naked eye directly observe the color of five parts of samples, judge the toxicity size of sample by contrasting the color distortion of various toxic solution and standard solution.Result is as shown in Figure 2, can be seen that, along with 3, the increase of 5-DCP concentration, color sample according to a, b, c, d and e order gradually become yellow by dark green, detection sample and control sample color are inconsistent, and water sample to be measured is poisonous, and toxicity size order is e > d > c > b.
Embodiment 2
Genetically engineered bacteria application in the total toxicity detection of water body:
From 30 DEG C, the cultivation temperature in embodiment 1 step 4 is become 37 DEG C, and other steps are identical with embodiment 1.Testing result is as shown in Figure 3, can be seen that, along with 3, the increase of 5-DCP concentration, color sample according to a, b, c, d and e order gradually become yellow by dark green, detection sample and control sample color are inconsistent, and water sample to be measured is poisonous, and toxicity size order is e > d > c > b.
Embodiment 3
Genetically engineered bacteria application in the total toxicity detection of water body:
From 15h, the incubation time in embodiment 1 step 4 is become 20h, and other steps are identical with embodiment 1.Testing result is as shown in Figure 4, can be seen that, along with 3, the increase of 5-DCP concentration, color sample according to a, b, c, d and e order gradually become yellow by dark green, detection sample and control sample color are inconsistent, and water sample to be measured is poisonous, and toxicity size order is e > d > c > b.
Embodiment 4
Genetically engineered bacteria application in the total toxicity detection of water body:
Four parts of culture medium of all the other in embodiment 1 step 4 are added volume is 2mL, and concentration is 25mg/L respectively, 50mg/L, 100mg/L, the Cu of 200mg/L2+, i.e. Cu in five parts of samples2+Concentration respectively (a) 0mg/L, (b) 2.5mg/L, (c) 5mg/L, (d) 10mg/L and (e) 20mg/L, other steps and embodiment 1 are identical.Testing result is as shown in Figure 5, it can be seen that except standard solution becomes bottle green, add Cu2+Each solution colour and the color of culture medium before cultivating there is no difference, it is seen that the method is to Cu2+Detection sensitiveer, add a small amount of Cu2+Just can the breeding of suppressor gene engineered bacterial, detection sample and control sample color are inconsistent, and water sample to be measured is poisonous.
Embodiment 5
Genetically engineered bacteria application in the total toxicity detection of water body:
Four parts of culture medium of all the other in embodiment 1 step 4 are added volume is 2mL, and concentration is 25mg/L respectively, 50mg/L, 100mg/L, the Zn of 200mg/L2+, i.e. Zn in five parts of samples2+Concentration respectively (a) 0mg/L, (b) 2.5mg/L, (c) 5mg/L, (d) 10mg/L and (e) 20mg/L, other steps and embodiment 1 are identical.Testing result is as shown in Figure 6, it can be seen that standard solution becomes bottle green, adds Zn2+Solution all become blackish green, but be as Zn2+The increase of concentration, color sample according to a, b, c, d and e order deepen gradually.Visible addition Zn2+Can promoting the breeding of genetically engineered bacteria and the secretion of electron mediator, detection sample and control sample color are inconsistent, and water sample to be measured is poisonous, and toxicity size order is e > d > c > b.
Embodiment 6
Genetically engineered bacteria application in the total toxicity detection of water body:
Embodiment 1 step 3 and step 4 are replaced by: in the super-clean bench of sterilizing, 1.8mL antibiotic after degerming is added in the culture medium after 178.2mL sterilizing, genetically engineered bacteria is inoculated into containing in antibiotic culture medium, after mixing, culture medium equal-volume is packed as ten parts (every part of 18mL), it is the deionized water of 2mL that a copy of it adds volume, all the other nine parts add volume is 2mL, concentration is (a) 0mg/L respectively, (b) 0.78mg/L, (c) 1.56mg/L, (d) 3.13mg/L, (e) 6.25mg/L, (f) 12.5mg/L, (g) 25mg/L, (h) 50mg/L, the Hg of (i) 100mg/L and (j) 200mg/L2+, in 30 DEG C of constant-temperature tables, cultivate 15h with 220rpm rotating speed.Other steps are identical with embodiment 1.Testing result is as shown in Figure 7, it can be seen that standard solution becomes bottle green, along with Hg2+The increase of concentration, color sample according to a, b, c, d, e, f, g, h, i and j order become the true qualities of culture medium gradually from bottle green, detection is limited to 6.25mg/L, detection sample and control sample color are inconsistent, water sample to be measured is poisonous, and toxicity size order is j > i > h > g > f > e > d > c > b.
Embodiment 7
Genetically engineered bacteria application in the total toxicity detection of water body:
All the other in embodiment 1 step 4 four parts are added volume is 2mL, and concentration is (b) 12.5mg/LCd respectively2+And 12.5mg/LCr3+, (c) 25mg/LCd2+And 25mg/LCr3+, (d) 50mg/LCd2+And 50mg/LCr3+, (e) 100mg/LCd2+And 100mg/LCr3+, other steps are identical with embodiment 1.Testing result is as shown in Figure 8, it can be seen that standard solution becomes bottle green, along with Cd2+And Cr3+The increase of mixture concentration, color sample according to a, b, c, d and e order become the true qualities of culture medium gradually from bottle green, detection sample and control sample color are inconsistent, water sample to be measured is poisonous, and toxicity size order is e > d > c > b.

Claims (8)

1. genetically engineered bacteria application in the total toxicity detection of water body, it is characterised in that step is as follows:
Step one, genetically engineered bacteria is inoculated into ensure that in the culture medium that this genetically engineered bacteria is normally bred, obtain postvaccinal culture medium；
Described genetically engineered bacteria can autocrine electron mediator；
Step 2, postvaccinal culture medium equal-volume being packed as two parts of samples, it is the deionized water of V that a sample adds volume, obtains control sample, and another part of sample adds the water sample to be measured that volume is V, obtains detection sample；
Or postvaccinal culture medium equal-volume is packed as the sample of more than three parts, and it is the deionized water of V that a sample adds volume, obtains control sample, remaining sample is separately added into the water sample to be measured of same concentrations that volume is V or variable concentrations, obtains detection sample；
Under step 3, same culture conditions, cultivate control sample and detection sample, observe control sample and the color of detection sample, if the detection sample after cultivating and control sample solid colour, then water sample to be measured is nontoxic, if the detection sample after cultivating is inconsistent with control sample color, then water sample to be measured is poisonous.
2. the genetically engineered bacteria according to claim 1 application in the total toxicity detection of water body, it is characterised in that described genetically engineered bacteria is obtained by rhlIR gene cluster process LAN in pseudomonas aeruginosa P.Aeruginosa.
3. the genetically engineered bacteria according to claim 2 application in the total toxicity detection of water body, it is characterized in that, in described step one, ensure the mixture of the antibiotic that culture medium is sterilizing that this genetically engineered bacteria normally breeds and the culture medium of sterilizing, described antibiotic is tetracycline, after mixing with the culture medium of sterilizing, final concentration of 100-200ug/mL, or it is tetracycline and ampicillin mixture, after mixing with the culture medium of sterilizing, the final concentration of 100-200ug/mL of tetracycline, the final concentration of 50-150ug/mL of ampicillin.
4. the genetically engineered bacteria according to claim 3 application in the total toxicity detection of water body, it is characterised in that described culture medium is the one in PB culture medium, M9 culture medium, LB culture medium, PYO culture medium.
5. the genetically engineered bacteria according to claim 3 application in the total toxicity detection of water body, it is characterised in that the culture medium of described sterilizing is that at 121 DEG C, under 1 atmospheric pressure after sterilizing 20min, cooling obtains by by ultrasonic for culture medium mixing, sealing.
6. the genetically engineered bacteria according to claim 3 application in the total toxicity detection of water body, it is characterised in that the antibiotic of described sterilizing is by being dissolved in deionized water by antibiotic, then obtains with the membrane filtration of 0.05～0.5 μm.
7. the genetically engineered bacteria according to claim 2 application in the total toxicity detection of water body, it is characterized in that, the condition of culture of described step 3 is: with the rotating speed of 100～250rpm, control sample and detection sample are cultivated 10～35h in the constant-temperature table of 10～45 DEG C.
8. the genetically engineered bacteria according to claim 7 application in the total toxicity detection of water body, it is characterised in that described incubation time is 15h, cultivation temperature is 30 DEG C.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113122609A (en) * 2021-04-27 2021-07-16 中国科学院长春应用化学研究所 Simple and rapid colorimetric detection method for total toxicity of water body
CN113122609B (en) * 2021-04-27 2023-06-13 中国科学院长春应用化学研究所 Simple and rapid colorimetric detection method for total toxicity of water body

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