CN113106045B - Construction and application of lead ion microorganism whole-cell biosensor taking water-soluble blue pigment as output signal - Google Patents

Abstract

The invention discloses construction and application of a lead ion microorganism whole-cell biosensor taking water-soluble blue pigment as an output signal. The invention provides a recombinant bacterium, which is obtained by introducing a recombinant vector A into a recipient bacterium; the recombinant vector A contains a DNA fragment A; the DNA fragment A contains a pbr bidirectional promoter, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side of the pbr bidirectional promoter is a water-soluble cyanine synthetic gene module. The recombinant strain can be used as a lead ion microorganism whole-cell biosensor taking water-soluble blue pigment as an output signal. On the one hand, qualitative indication of target heavy metal exposure can be achieved by color change, and on the other hand, quantitative analysis of target heavy metals can be performed by colorimetric methods.

Description

Construction and application of lead ion microorganism whole-cell biosensor taking water-soluble blue pigment as output signal

Technical Field

The invention relates to the technical field of biology, in particular to construction and application of a lead ion microorganism whole-cell biosensor taking water-soluble blue pigment as an output signal.

Background

The metal elements widely exist in nature in natural abundance, and include transition metals, some metalloids, lanthanides, actinides and the like, wherein some elements (such as Fe, ca, co, cu, mn, zn, mg and the like) have very important functions in the life process, and the elements are widely involved in various aspects of cell growth and development, gene transcription regulation, nerve signal transmission and the like. On the other hand, however, some other elements (e.g., hg, pb, cd, as, cr, etc.) among the metal elements are extremely toxic to organisms at a very low concentration, and unlike organic compounds, heavy metals have an accumulation property and are hardly degraded in the natural environment.

At present, the method for detecting the heavy metal pollutants in the environment is mainly a physicochemical method. Such as by atomic spectrometry, inductively coupled plasma mass spectrometry, and the like. The physicochemical detection method needs expensive professional equipment, different pretreatment methods need to be searched according to the properties of the sample, the detection timeliness is poor, and the requirement on the professional technology of operators is high. The advent of the microbial sensor provides more choices for the detection of heavy metal pollutants in the environment, and the microbial sensor mainly detects bioavailable heavy metal poisons, so that the detection is rapid and the cost is low.

Indigo is a natural water-soluble blue pigment synthesized by various microorganisms, and the biosynthesis pathway of indigo is well studied. The natural pigment synthetic gene cluster is used as a reporter gene module, and is expected to realize the biological sensing of heavy metals based on color change and the naked eye identification of the exposure of toxic heavy metals. Relevant researches are rarely reported.

At present, no report related to a lead ion microorganism whole cell biosensor taking water-soluble cyanine indigo as an output signal exists.

Disclosure of Invention

The invention claims construction and application of a lead ion microorganism whole cell biosensor taking water-soluble blue pigment indigo as an output signal.

In a first aspect, the invention claims a recombinant bacterium.

The recombinant bacterium claimed by the invention is obtained by introducing the recombinant vector A into a recipient bacterium.

The recombinant vector A contains a DNA fragment A.

The DNA fragment A contains a pbr bidirectional promoter, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side of the pbr bidirectional promoter is a water-soluble blue pigment indigo synthetic gene module.

The water-soluble blue pigment indigo synthesis gene module is assembled in a bicistronic form and encodes BspA protein and PcpS protein.

The nucleotide sequence of the pbr bidirectional promoter is 442 th to 526 th positions of SEQ ID No.1.

The amino acid sequence of the PbrR protein is shown as SEQ ID No. 2.

The amino acid sequence of the BspA protein is shown in SEQ ID No. 3.

The amino acid sequence of the PcpS protein is shown as SEQ ID No. 4.

The nucleotide sequence of the coding gene of the PbrR protein is a reverse complementary sequence of 7 th to 441 th of SEQ ID No.1.

The nucleotide sequence of the coding gene of the BspA protein is 569-4417 th site of SEQ ID No.1.

The nucleotide sequence of the coding gene of the PcpS protein is 4435-5163 of SEQ ID No.1.

Further, the nucleotide sequence of the water-soluble blue pigment indigo synthesis gene module is 569-5163 of SEQ ID No.1.

Furthermore, the nucleotide sequence of the DNA fragment A is SEQ ID No.1.

The recombinant vector A is obtained by replacing a small fragment between enzyme cutting sites BglII and SacI of pET-21a (+) plasmid with the DNA fragment A.

In the present invention, the recipient bacterium is Escherichia coli.

Further, the Escherichia coli is Escherichia coli TOP10.

In a second aspect, the invention claims application of the recombinant bacterium in the first aspect in serving as or preparing a lead ion microorganism whole-cell biosensor taking water-soluble blue pigment indigo as an output signal.

In a third aspect, the invention claims any of the following products or applications:

p1, recombinant vector a as described in the previous first aspect;

p2, DNA fragment a as described in the first aspect hereinbefore;

the use of the DNA fragment A of the recombinant vector A or P2 of P3 or P1 for the preparation of the recombinant bacteria of the first aspect;

p4, a kit comprising a recombinant bacterium as described in the first aspect hereinbefore and lead ions as a standard;

use of the kit of parts of the DNA fragment A or P4 of the recombinant vector A or P2 of P5 or P1 or the recombinant bacterium of the first aspect in the detection of lead ions.

Further, the lead ion detection is the qualitative and/or quantitative detection of lead ions on the liquid sample. Wherein the kit can be used for quantitative detection.

Wherein the liquid sample satisfies the following conditions: and (3) placing the recombinant bacteria in the liquid sample for 3h, wherein the growth and the propagation of the recombinant bacteria can be normally carried out. For example, 9 volumes of the sample to be tested and 1 volume of 10 times the concentrated LB liquid medium were mixed to obtain a sample.

In an embodiment of the present invention, the liquid sample is a culture medium for culturing the recombinant bacteria. In particular to a liquid LB culture medium containing 50mg/L ampicillin.

In a fourth aspect, the invention claims any of the following methods:

the method comprises the following steps: a method for detecting whether a liquid sample contains lead ions is the following method A or method B:

the method A comprises the following steps: the bacterial liquid observation method comprises the following steps: placing the recombinant bacterium in the first aspect into the liquid sample to be tested, performing shake culture at 250rpm at 25-37 ℃ (such as 37 ℃) for 3h, observing the color change of the liquid sample to be tested, and if the color reaction of water-soluble blue pigment indigo is observed, determining that the liquid sample to be tested contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain lead ions.

The method B comprises the following steps: an A600 value assay comprising the steps of: placing the recombinant bacteria in the first aspect into the liquid sample to be tested, shake-culturing at 25-37 ℃ (e.g. 37 ℃) and 250rpm for 3h, centrifuging (e.g. 3500rpm for 5 min), taking the supernatant, and determining the A600 value, which is referred to as the A600 value of the liquid sample group to be tested for short; if the A600 value of the liquid sample group to be detected is significantly larger than the A600 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or is candidate to contain lead ions.

And the determination method of the A600 value of the control group is different from the determination method of the A600 value of the liquid sample group to be tested only in that the liquid sample to be tested is replaced by a liquid sample without lead ions. The liquid sample that does not contain lead ions preferably differs from the liquid sample to be tested only in that it does not contain lead ions.

Further, the method is suitable for the case where the liquid sample to be tested contains lead ions of 0.033 μ M or more (e.g., 0.26 μ M or more, further e.g., 0.26-8.3 μ M, further e.g., 4.17 μ M or more).

When the liquid sample to be detected is colorless or can be distinguished from the color of the liquid sample to be detected (visually distinguished) by superimposing the water-soluble blue pigment indigo for color development, the result is preferably determined by a visual observation method (that is, the method A is preferably applicable to the case that the liquid sample to be detected contains lead ions of more than 4.17 mu M). When the liquid sample to be detected is difficult to distinguish from the self color (can not be distinguished by naked eyes) after being colored by superposing the water-soluble blue pigment indigo, the method preferably adopts an A600 value determination method (namely, the method B is preferably suitable for the condition that the liquid sample to be detected contains more than 0.033 mu M lead ions) to carry out result judgment.

The second method comprises the following steps: a method for detecting the content of lead ions in a liquid sample comprises the following steps:

(A1) Placing the recombinant strain in the first aspect into a series of lead ion liquid samples with known concentration, performing shake culture at 250rpm for 3h at 25-37 ℃ (such as 37 ℃), centrifuging (such as centrifuging at 3500rpm for 5 min), taking supernatant, determining A600 value, and then drawing a standard curve according to the lead ion concentration and the A600 value;

(A2) And (3) replacing the series of lead ion liquid samples with known concentrations in the step (A1) with the liquid sample to be detected, repeating the step (A1) to obtain an A600 value of the liquid sample to be detected, and substituting the value into the standard curve to obtain the lead ion content in the liquid sample to be detected.

Further, the second method is suitable for the case that the liquid sample to be tested contains 0.26 μ M to 8.3 μ M of lead ions.

In the above two methods, the recombinant bacterium is in the liquid sample to be tested or the series of known bacteriaThe content of the lead ion liquid sample with concentration is OD ₆₀₀ ＝0.6。

In the above aspects, the lead ion is a divalent lead ion (Pb (II)).

In each of the above aspects, the lead ions are preferably present in the form of a soluble divalent lead salt, such as lead acetate, lead chloride, lead nitrate, and the like.

In a particular embodiment of the invention, the divalent lead ion (Pb (II)) is present in particular in the form of lead acetate.

According to the invention, a gene module for activating the synthesis pathway of heavy metal lead ion activated indinoid pigment is built through a synthetic biology means, so that the expression of target heavy metal lead induced indinoid (extracellular secretion, blue culture solution) is realized, and concept verification is carried out for the assumption that natural pigment heterologous biosynthesis indicates heavy metal lead exposure. On the one hand, the color change can realize qualitative indication of target heavy metal exposure, and on the other hand, the target heavy metal can be quantitatively analyzed through colorimetry.

Drawings

FIG. 1 is a molecular mechanical diagram of a lead ion microorganism whole-cell biosensor using water-soluble blue pigment indigo as an output signal according to the present invention.

FIG. 2 shows the identification of recombinant bacterium TOP10/pPpbr-ind, specifically the identification result of BglII and XbaI double digestion of the objective plasmid extracted therefrom.

FIG. 3 shows the response results of the recombinant strain TOP10/pPpbr-ind to different concentrations of lead ions (A600 detection method).

FIG. 4 shows the response results of the recombinant strain TOP10/pPpbr-ind to different concentrations of lead ions (visual observation).

FIG. 5 shows the response results of the recombinant strain TOP10/pPpbr-ind to different metal ions. The upper panel is the various metal induced group supernatants A600, and the lower panel is a representative photograph.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 construction and application of lead ion microbial whole-cell biosensor using water-soluble blue pigment indigo as output signal

1. Construction of recombinant plasmid pPpbr-ind

First, a water-soluble blue pigment indigo synthetic gene module (bspA-pcpS bicistron, shown as positions 569-5163 of SEQ ID No. 1) is synthesized by using pET-21a (+) as a vector and is inserted into pET-21a (+) by utilizing NdeI/SacI, and the recombinant plasmid which is verified to be correct through sequencing is named as pT7lac-ind.

The structure of pT7lac-ind is described as: a recombinant plasmid in which a small segment between NdeI/SacI of a pET-21a (+) vector is replaced by a DNA segment shown in positions 569-5163 of SEQ ID No.1.

Secondly, a pbrR-pbr promoter gene module (7 th to 526 th positions of SEQ ID No. 1) is synthesized by the whole gene, is inserted into a pT7lac-ind vector by BglII/XbaI, and the recombinant plasmid after being verified to be correct by sequencing is named as pPpbr-ind.

The structure of pPppbr-ind is described as: the small fragment between BglII/SacI of pET-21a (+) vector was replaced with a recombinant plasmid of the DNA fragment shown in SEQ ID No.1.

The 7 th to 526 th positions of SEQ ID No.1 are PbrR-pbr bidirectional promoter sequence and are divalent lead ion Pb (II) sensing elements, the 442 th to 526 th positions of SEQ ID No.1 are nucleotide sequences of pbr bidirectional promoter, and the 7 th to 441 th positions of SEQ ID No.1 are reverse complementary sequences of pbrR gene and encode PbrR protein shown in SEQ ID No. 2.

The 569-5163 th site of SEQ ID No.1 is bspA-pcpS, an indinoid synthetic gene cluster which is assembled in a bicistronic form and encodes 2 proteins. The 569-4417 th site of SEQ ID No.1 is a bspA gene which encodes the BspA protein shown in SEQ ID No. 3. The 4435-5163 position of SEQ ID No.1 is pcpS gene, which encodes the pcpS protein shown in SEQ ID No. 4.

When lead ions appear, 2 genes are activated to be coupled and transcribed, 2 enzymes generated by translation take glutamine as a substrate, and the individine is catalytically generated. The molecular mechanism is shown in FIG. 1.

2. Construction of recombinant bacterium TOP10/pPpbr-ind

And (3) transforming the recombinant plasmid pPspbr-ind constructed in the step one into Escherichia coli TOP10 to obtain a recombinant strain TOP 10/pPspbr-ind.

The target plasmid is extracted from the recombinant TOP10/pPpbr-ind and subjected to BglII and XbaI double enzyme digestion, and the result is shown in figure 2. As can be seen, two bands of approximately 9.9K and 532bp were obtained, consistent with expectations. The correctness is further confirmed by sequencing verification.

3. Response of recombinant bacterium TOP10/pPpbr-ind to lead ions with different concentrations

1. Inoculating the recombinant bacterium TOP10/pPpbr-ind constructed in the step two into a liquid LB culture medium containing 50mg/L ampicillin, and carrying out shake culture at 37 ℃ and 250rpm until OD is reached _600nm The value =0.6.

2. Lead acetate was added to final concentrations of 0, 0.008, 0.016, 0.033, 0.065, 0.13, 0.26, 0.52, 1.04, 2.08, 4.17, 8.3, 16.7, 33.3, 66.7, 133.4, 266.8. Mu. Mol/L Pb (II), respectively, and cultured at 37 ℃ and 250rpm for 3 hours with shaking.

3. The bacterial liquid of each group is collected, 3500rpm is carried out, and centrifugation is carried out for 5min.

4. mu.L of the culture supernatant was aspirated, placed in a 96-well plate, and absorbance at 600nm (A600) was measured.

The results are shown in table 1 and fig. 3 and 4.

In FIG. 3, a is A600 of the supernatant of Pb (II) -induced bacterial liquid at 0, 0.008, 0.016, 0.033, 0.065, 0.13, 0.26, 0.52, 1.04, 2.08, 4.17, 8.3, 16.7, 33.3, 66.7, 133.4, 266.8. Mu. Mol/L; b is an A600 of the supernatant of the Pb (II) induced bacteria liquid with the concentration of 0-16.7 mu mol/L enlarged at the red square of the picture a; c is an enlargement at a red box of the b picture, 0.26, 0.52, 1.04, 2.08, 4.17 and 8.3 mu mol/L Pb (II) induce A600 of the bacterial liquid supernatant, and the lead concentration and A600 are in a linear relation in the range; d is 0, 0.008, 0.016, 0.033, 0.065, 0.13, 0.26, 0.52, 1.04 and 2.08 mu mol/L Pb (II) induced bacteria supernatant A600, and the histogram shows that the 0.033 mu mol/L Pb (II) induced group is statistically different from the 0 mu mol/L Pb (II) group. Namely, the detection limit of the biosensor is 0.033 mu mol/L Pb (II).

Photographs of the supernatants are shown in FIG. 4, where the limits of the instrumental and visual detection are indicated.

Table 1, detection results of absorbance at 600nm for each group

Pb(II)μM	A600	SD
				0	0.05	0.003
0.008	0.051	0.003
			0.016	0.053	0.003
0.033	0.056	0.004
			0.065	0.061	0.003
0.13	0.067	0.008
			0.26	0.081	0.012
0.52	0.094	0.011
			1.04	0.103	0.016
2.08	0.123	0.018
			4.17	0.189	0.016
8.3	0.3	0.025
			16.7	0.414	0.027
33.3	0.468	0.032
			66.7	0.49	0.038
133.4	0.499	0.036
			266.8	0.506	0.042

3. Response of recombinant bacterium TOP10/pPpbr-ind to different metal ions

1. Inoculating the recombinant bacterium TOP10/pPpbr-ind constructed in the step two into a liquid LB culture medium containing 50mg/L ampicillin, and carrying out shake culture at 37 ℃ and 250rpm until OD is reached _600nm Value =0.6.

2. The final concentration was 30. Mu.M of Cu (II), cr (III), ni (II), mn (II), pb (II), zn (II), cd (II) or Hg (II) added to the above culture, and cultured at 37 ℃ and 250rpm for 3 hours with shaking.

3. The bacterial liquid of each group is collected, 3500rpm, and 5min of centrifugation is carried out.

4. 200. Mu.L of the culture supernatant was aspirated, and placed in a 96-well plate to measure absorbance at 600 nm.

The results are shown in table 2 and fig. 5. It can be seen that the Pb (II) exposed group supernatant appeared blue, with a600 value significantly higher than the other metal groups.

Table 2, results of detection of absorbance at 600nm for each group

Metal species	A600	SD
			Cu(II)	0.048	0.011
Cr(III)	0.052	0.012
			Ni(II)	0.075	0.016
Mn(II)	0.056	0.013
			Pb(II)	0.477	0.016
Zn(II)	0.053	0.015
			Cd(II)	0.072	0.015
Hg(II)	0.078	0.015

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> Shenzhen market occupational disease prevention and treatment hospital

<120> construction and application of lead ion microorganism whole cell biosensor using water-soluble blue pigment as output signal

<130> GNCLN210753

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 5169

<212> DNA

<213> Artificial sequence

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ctccagcagc gtgatgacct ccccacagtc ctgcatcggg ttgtctcgca gacccagcaa 240

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ctcaatgtgc gcctcaccgt acaaccgaaa gttgccaccg cttcgcgctg gctttggcag 360

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cgtcaggcgg agttcctggc gggtcgtctg tgcgcgcgtg cggcgctgtt cgcgctggat 4680

ggtcgtgcgc agactccggc tgttggcgaa gatcgtgcgc cggtttggcc ggcggcgatc 4740

tccggctcta tcacccacgg cgatcgctgg gcggcggcgc tggttgcggc gcgcggcgat 4800

tggcgtggtc tgggtctgga tgttgaaacc ctgctggaag cggaacgtgc gcgttacctg 4860

cacggcgaaa tcctgaccga aggcgaacgt ctgcgtttcg cggatgatct ggaacgtcgt 4920

accggcctgc tggtgaccct ggcgttctcc ctgaaagaaa gcctgttcaa agcgctgtac 4980

ccgctggttg gcaaacgttt ctacttcgaa cacgcagaac tgctggaatg gcgtgcggac 5040

ggtcaagcgc gtctgcgtct gctgaccgat ctgagcccgg aatggcgtca cggttctgaa 5100

ctggatgcgc agttcgcggt tctggacggc cgtctgctgt ctctggttgc ggttggtgcg 5160

taagagctc 5169

<210> 2

<211> 144

<212> PRT

<213> Artificial sequence

<400> 2

Met Glu Ile Arg Ile Gly Asp Leu Ala Lys Arg Ser Gly Cys Glu Val

1 5 10 15

Val Thr Ile Arg Tyr Tyr Glu Lys Glu Gly Leu Leu Pro Lys Pro Ala

20 25 30

Arg Ser Gly Gly Asn Phe Arg Leu Tyr Gly Glu Ala His Ile Glu Arg

35 40 45

Leu Gln Phe Ile Arg His Cys Arg Ser Leu Asp Met Thr Leu Ser Glu

50 55 60

Ile Arg Ala Leu Leu Gly Leu Arg Asp Asn Pro Met Gln Asp Cys Gly

65 70 75 80

Glu Val Ile Thr Leu Leu Glu Ala His Ile Gln Gln Val Glu Met Arg

85 90 95

Val Ser Ala Leu Leu Gln Leu Lys Arg His Leu Val Asp Leu Arg Glu

100 105 110

Lys Cys Ser Gly Ser Arg Ser Val Glu Ala Cys Gly Ile Leu Gln Gly

115 120 125

Leu Gly Asn Cys His Gly Glu Ser Ala Thr Asn Ser Gln Thr Ser Gly

130 135 140

<210> 3

<211> 1282

<212> PRT

<213> Artificial sequence

<400> 3

Met Thr Leu Gln Glu Thr Ser Val Leu Glu Pro Thr Leu Gln Gly Thr

1 5 10 15

Thr Thr Leu Pro Gly Leu Leu Ala Gln Arg Val Ala Glu His Pro Glu

20 25 30

Ala Ile Ala Val Ala Tyr Arg Asp Asp Lys Leu Thr Phe Arg Glu Leu

35 40 45

Ala Ser Arg Ser Ala Ala Leu Ala Asp Tyr Leu Glu His Leu Gly Val

50 55 60

Ser Ala Asp Asp Cys Val Gly Leu Phe Val Glu Pro Ser Ile Asp Leu

65 70 75 80

Met Val Gly Ala Trp Gly Ile Leu Asn Ala Gly Ala Ala Tyr Leu Pro

85 90 95

Leu Ser Pro Glu Tyr Pro Glu Asp Arg Leu Arg Tyr Met Ile Glu Asn

100 105 110

Ser Glu Thr Lys Ile Ile Leu Ala Gln Gln Arg Leu Val Ser Arg Leu

115 120 125

Arg Glu Leu Ala Pro Lys Asp Val Thr Ile Val Thr Leu Arg Glu Ser

130 135 140

Glu Ala Phe Val Arg Pro Glu Gly Thr Glu Ala Pro Ala Ala Arg Ser

145 150 155 160

Ala Arg Pro Asp Thr Leu Ala Tyr Val Ile Tyr Thr Ser Gly Ser Thr

165 170 175

Gly Lys Pro Lys Gly Val Met Ile Glu His Arg Ser Ile Val Asn Gln

180 185 190

Leu Gly Trp Leu Arg Glu Thr Tyr Ala Ile Asp Arg Ser Lys Val Ile

195 200 205

Leu Gln Lys Thr Pro Met Ser Phe Asp Ala Ala Gln Trp Glu Ile Leu

210 215 220

Ser Pro Ala Asn Gly Ala Thr Val Val Met Gly Ala Pro Gly Val Tyr

225 230 235 240

Ala Asp Pro Glu Gly Leu Ile Glu Thr Ile Val Lys His Asn Val Thr

245 250 255

Thr Leu Gln Cys Val Pro Thr Leu Leu Gln Gly Leu Ile Asp Thr Glu

260 265 270

Lys Phe Pro Glu Cys Val Ser Leu Gln Gln Ile Phe Ser Gly Gly Glu

275 280 285

Ala Leu Ser Arg Leu Leu Ala Ile Gln Thr Thr Gln Glu Met Pro Gly

290 295 300

Arg Ala Leu Ile Asn Val Tyr Gly Pro Thr Glu Thr Thr Ile Asn Ser

305 310 315 320

Ser Ser Phe Pro Val Asp Pro Ala Asp Leu Asp Glu Gly Pro Gln Ser

325 330 335

Ile Ser Ile Gly Ser Pro Val His Gly Thr Thr Tyr His Ile Leu Asp

340 345 350

Lys Glu Thr Leu Lys Pro Val Gly Val Gly Glu Ile Gly Glu Leu Tyr

355 360 365

Ile Gly Gly Ile Gln Leu Ala Arg Gly Tyr Leu His Arg Asp Asp Leu

370 375 380

Thr Ala Glu Arg Phe Leu Glu Ile Glu Leu Glu Glu Gly Ala Glu Pro

385 390 395 400

Val Arg Leu Tyr Lys Thr Gly Asp Leu Gly Gln Trp Asn Asn Asp Gly

405 410 415

Thr Val Gln Phe Ala Gly Arg Ala Asp Asn Gln Val Lys Leu Arg Gly

420 425 430

Tyr Arg Val Glu Leu Asp Glu Ile Ser Leu Ala Ile Glu Asn His Asp

435 440 445

Trp Val Arg Asn Ala Ala Val Ile Val Lys Asn Asp Gly Arg Thr Gly

450 455 460

Phe Gln Asn Leu Ile Ala Cys Ile Glu Leu Ser Glu Lys Glu Ala Ala

465 470 475 480

Leu Met Asp Gln Gly Asn His Gly Ser His His Ala Ser Lys Lys Ser

485 490 495

Lys Leu Gln Val Lys Ala Gln Leu Ser Asn Pro Gly Leu Arg Asp Asp

500 505 510

Ala Glu Leu Ala Ala Arg Pro Ala Phe Asp Leu Glu Gly Ala Glu Pro

515 520 525

Thr Pro Glu Gln Arg Ala Arg Val Phe Ala Arg Lys Thr Tyr Arg Phe

530 535 540

Tyr Glu Gly Gly Ala Val Thr Gln Ala Asp Leu Leu Gly Leu Leu Gly

545 550 555 560

Ala Thr Val Thr Ala Gly Tyr Ser Arg Lys Ala Ala Asp Leu Ala Pro

565 570 575

Ala Glu Leu Gly Gln Ile Leu Arg Trp Phe Gly Gln Tyr Ile Ser Glu

580 585 590

Glu Arg Leu Leu Pro Lys Tyr Gly Tyr Ala Ser Pro Gly Ala Leu Tyr

595 600 605

Ala Thr Gln Met Tyr Phe Glu Leu Glu Gly Val Gly Gly Leu Lys Pro

610 615 620

Gly Tyr Tyr Tyr Tyr Gln Pro Val Arg His Gln Leu Val Leu Ile Ser

625 630 635 640

Glu Arg Glu Ala Thr Gly Lys Ala Thr Ala Gln Ile His Phe Ile Gly

645 650 655

Lys Lys Ser Gly Ile Glu Pro Val Tyr Lys Asn Asn Ile Leu Glu Val

660 665 670

Leu Glu Ile Glu Thr Gly His Met Val Gly Leu Phe Glu Gln Ile Leu

675 680 685

Pro Ala Tyr Gly Leu Asp Ile His Asp Arg Ala Tyr Glu Pro Ala Val

690 695 700

Lys Asp Leu Leu Asp Val Ala Asp Glu Asp Tyr Tyr Leu Gly Thr Phe

705 710 715 720

Glu Leu Val Pro His Ala Gly Ala Arg Asp Asp Gln Ala Glu Val Tyr

725 730 735

Val Gln Thr His Gly Gly Lys Val Ala Gly Leu Pro Glu Gly Gln Tyr

740 745 750

Arg Tyr Glu Asn Gly Glu Leu Thr Arg Phe Ser Asp Asp Ile Val Leu

755 760 765

Lys Lys His Val Ile Ala Ile Asn Gln Ser Val Tyr Gln Ala Ala Ser

770 775 780

Phe Gly Ile Ser Val Tyr Ser Arg Ala Glu Glu Glu Trp Leu Lys Tyr

785 790 795 800

Ile Thr Leu Gly Lys Lys Leu Gln His Leu Met Met Asn Gly Leu Asn

805 810 815

Leu Gly Phe Met Ser Ser Gly Tyr Ser Ser Lys Thr Gly Asn Pro Leu

820 825 830

Pro Ala Ser Arg Arg Met Asp Ala Val Leu Gly Ala Asn Gly Val Asp

835 840 845

Ser Ala Pro Met Tyr Phe Phe Val Gly Gly Arg Ile Ser Asp Glu Gln

850 855 860

Ile Gly His Glu Gly Met Arg Glu Asp Ser Val His Met Arg Gly Pro

865 870 875 880

Ala Glu Leu Ile Arg Asp Asp Leu Val Ser Phe Leu Pro Asp Tyr Met

885 890 895

Ile Pro Asn Arg Val Val Val Phe Asp Arg Leu Pro Leu Ser Ala Asn

900 905 910

Gly Lys Ile Asp Val Lys Ala Leu Ala Ala Ser Asp Gln Val Asn Ala

915 920 925

Glu Leu Val Glu Arg Pro Phe Val Ala Pro Arg Thr Glu Thr Glu Lys

930 935 940

Glu Ile Ala Ala Val Trp Glu Lys Ala Leu Arg Arg Glu Asn Ala Ser

945 950 955 960

Val Gln Asp Asp Phe Phe Glu Ser Gly Gly Asn Ser Leu Ile Ala Val

965 970 975

Gly Leu Val Arg Glu Leu Asn Ala Arg Leu Gly Val Ser Leu Pro Leu

980 985 990

Gln Ser Val Leu Glu Ser Pro Thr Ile Glu Lys Leu Ala Arg Arg Leu

995 1000 1005

Glu Arg Glu Val Ala Gln Glu Ser Ser Arg Phe Val Arg Leu His

1010 1015 1020

Ala Glu Thr Gly Lys Ala Arg Pro Val Ile Cys Trp Pro Gly Leu

1025 1030 1035

Gly Gly Tyr Pro Met Asn Leu Arg Ser Leu Ala Gly Glu Ile Gly

1040 1045 1050

Leu Gly Arg Ser Phe Tyr Gly Val Gln Ser Tyr Gly Ile Asn Glu

1055 1060 1065

Gly Glu Thr Pro Tyr Glu Thr Ile Thr Glu Met Ala Lys Lys Asp

1070 1075 1080

Ile Glu Ala Leu Lys Glu Ile Gln Pro Ala Gly Pro Tyr Thr Leu

1085 1090 1095

Trp Gly Tyr Ser Phe Gly Ala Arg Val Ala Phe Glu Thr Ala Tyr

1100 1105 1110

Gln Leu Glu Gln Ala Gly Glu Lys Val Asp Asn Leu Phe Leu Ile

1115 1120 1125

Ala Pro Gly Ser Pro Lys Val Arg Ala Glu Asn Gly Lys Val Trp

1130 1135 1140

Gly Arg Glu Ala Ser Phe Ala Asn Arg Gly Tyr Thr Thr Ile Leu

1145 1150 1155

Phe Ser Val Phe Thr Gly Thr Ile Ser Gly Pro Asp Leu Asp Arg

1160 1165 1170

Cys Leu Glu Thr Val Thr Asp Glu Ala Ser Phe Ala Glu Phe Ile

1175 1180 1185

Ser Glu Leu Lys Gly Ile Asp Val Asp Leu Ala Arg Arg Ile Ile

1190 1195 1200

Ser Val Val Gly Gln Thr Tyr Glu Phe Glu Tyr Ser Phe His Glu

1205 1210 1215

Leu Ala Glu Arg Thr Leu Gln Ala Pro Ile Ser Ile Phe Lys Ala

1220 1225 1230

Val Gly Asp Asp Tyr Ser Phe Leu Glu Asn Ser Ser Gly Tyr Ser

1235 1240 1245

Ala Glu Pro Pro Thr Val Ile Asp Leu Asp Ala Asp His Tyr Ser

1250 1255 1260

Leu Leu Arg Glu Asp Ile Gly Glu Leu Val Lys His Ile Arg Tyr

1265 1270 1275

Leu Leu Gly Glu

1280

<210> 4

<211> 242

<212> PRT

<213> Artificial sequence

<400> 4

Met Arg Ala Met Asn Asp Arg Leu Pro Ser Phe Cys Thr Pro Leu Asp

1 5 10 15

Asp Arg Trp Pro Leu Pro Val Ala Leu Pro Gly Val Gln Leu Arg Ser

20 25 30

Thr Arg Phe Asp Pro Ala Leu Leu Gln Pro Gly Asp Phe Ala Leu Ala

35 40 45

Gly Ile Gln Pro Pro Ala Asn Ile Leu Arg Ala Val Ala Lys Arg Gln

50 55 60

Ala Glu Phe Leu Ala Gly Arg Leu Cys Ala Arg Ala Ala Leu Phe Ala

65 70 75 80

Leu Asp Gly Arg Ala Gln Thr Pro Ala Val Gly Glu Asp Arg Ala Pro

85 90 95

Val Trp Pro Ala Ala Ile Ser Gly Ser Ile Thr His Gly Asp Arg Trp

100 105 110

Ala Ala Ala Leu Val Ala Ala Arg Gly Asp Trp Arg Gly Leu Gly Leu

115 120 125

Asp Val Glu Thr Leu Leu Glu Ala Glu Arg Ala Arg Tyr Leu His Gly

130 135 140

Glu Ile Leu Thr Glu Gly Glu Arg Leu Arg Phe Ala Asp Asp Leu Glu

145 150 155 160

Arg Arg Thr Gly Leu Leu Val Thr Leu Ala Phe Ser Leu Lys Glu Ser

165 170 175

Leu Phe Lys Ala Leu Tyr Pro Leu Val Gly Lys Arg Phe Tyr Phe Glu

180 185 190

His Ala Glu Leu Leu Glu Trp Arg Ala Asp Gly Gln Ala Arg Leu Arg

195 200 205

Leu Leu Thr Asp Leu Ser Pro Glu Trp Arg His Gly Ser Glu Leu Asp

210 215 220

Ala Gln Phe Ala Val Leu Asp Gly Arg Leu Leu Ser Leu Val Ala Val

225 230 235 240

Gly Ala

Claims (15)

1. The recombinant bacterium is obtained by introducing a recombinant vector A into a recipient bacterium;

the recombinant vector A contains a DNA fragment A;

the DNA fragment A contains a pbr bidirectional promoter, one side of the pbr bidirectional promoter is a PbrR protein coding gene, and the other side of the pbr bidirectional promoter is a water-soluble cyanine synthesis gene module;

the water-soluble blue pigment synthesis gene module is assembled in a bicistronic form and encodes BspA protein and PcpS protein;

the nucleotide sequence of the pbr bidirectional promoter is 442 th to 526 th positions of SEQ ID No. 1;

the amino acid sequence of the PbrR protein is shown as SEQ ID No. 2;

the amino acid sequence of the BspA protein is shown as SEQ ID No. 3;

the amino acid sequence of the PcpS protein is shown as SEQ ID No. 4;

the recipient bacterium is Escherichia coli.

2. The recombinant bacterium according to claim 1, wherein: the nucleotide sequence of the coding gene of the PbrR protein is a reverse complementary sequence of the 7 th to 441 th positions of SEQ ID No. 1;

the nucleotide sequence of the coding gene of the BspA protein is 569-4417 th site of SEQ ID No. 1;

the nucleotide sequence of the coding gene of the PcpS protein is 4435-5163 of SEQ ID No.1.

3. The recombinant bacterium according to claim 2, wherein: the nucleotide sequence of the water-soluble blue pigment synthetic gene module is 569-5163 th bit of SEQ ID No.1.

4. The recombinant bacterium according to claim 3, wherein: the nucleotide sequence of the DNA fragment A is SEQ ID No.1.

5. The recombinant bacterium according to claim 1, wherein: the recombinant vector A is the restriction enzyme cutting site of the DNA fragment A replacing pET-21a (+) plasmidBglII andSacsmall fragments between I.

6. The recombinant bacterium according to claim 1, wherein: the Escherichia coli is Escherichia coli TOP10.

7. The use of the recombinant strain of any one of claims 1 to 6 as or in the preparation of a lead ion microbial whole cell biosensor using a water-soluble blue pigment as an output signal.

8. Use according to claim 7, characterized in that: the lead ions are divalent lead ions.

9. A kit comprising the recombinant bacterium of any one of claims 1 to 6 and lead ions as a standard.

10. Use of the kit of claim 9 or the recombinant bacterium of any one of claims 1 to 6 for detecting lead ions.

11. Use according to claim 10, characterized in that: the lead ion detection is the qualitative and/or quantitative detection of lead ions on the liquid sample.

12. Use according to claim 10, characterized in that: the lead ions are divalent lead ions.

13. A method for detecting whether a liquid sample contains lead ions is a method A or a method B:

the method A comprises the following steps: the bacterial liquid observation method comprises the following steps: placing the recombinant bacterium of any one of claims 1-6 in a liquid sample to be tested, performing shake culture at 25-37 ℃ for 3h, observing the color change of the liquid sample to be tested, and if the color reaction of water-soluble blue pigment is observed, determining that the liquid sample to be tested contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions;

the method B comprises the following steps: an A600 value assay comprising the steps of: placing the recombinant bacterium of any one of claims 1-6 in a liquid sample to be tested, performing shake culture at 25-37 ℃ for 3h, centrifuging, taking supernatant, and determining an A600 value, namely the A600 value of the liquid sample group to be tested; if the A600 value of the liquid sample group to be detected is obviously greater than the A600 value of the control group, the liquid sample to be detected contains or is candidate to contain lead ions; otherwise, the liquid sample to be detected does not contain or candidate does not contain lead ions; and the determination method of the A600 value of the control group is different from the determination method of the A600 value of the liquid sample group to be tested only in that the liquid sample to be tested is replaced by a liquid sample without lead ions.

14. A method for detecting the content of lead ions in a liquid sample comprises the following steps:

(A1) Placing the recombinant bacteria of any one of claims 1-6 in a series of lead ion liquid samples with known concentration, performing shake culture at 25-37 ℃ for 3h, centrifuging, taking supernate, determining the A600 value, and then drawing a standard curve according to the lead ion concentration and the A600 value;

(A2) And (3) replacing the series of lead ion liquid samples with known concentrations in the step (A1) with the liquid sample to be detected, repeating the step (A1) to obtain an A600 value of the liquid sample to be detected, and substituting the value into the standard curve to obtain the lead ion content in the liquid sample to be detected.

15. The method according to claim 13 or 14, characterized in that: the lead ions are divalent lead ions.

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