WO2019061381A1

WO2019061381A1 - Class of arsenite inhibitory factor reporter gene plasmids, construction method therefor and application thereof

Info

Publication number: WO2019061381A1
Application number: PCT/CN2017/104721
Authority: WO
Inventors: 李先强; 方云; 姜昕; 许玫英; 郭俊
Original assignee: 广东省微生物研究所（广东省微生物分析检测中心）; 圣格诺斯有限公司
Priority date: 2017-09-30
Filing date: 2017-09-30
Publication date: 2019-04-04
Also published as: CN108064300B; CN108064300A

Abstract

Provided are a class of arsenite inhibitory factor reporter gene plasmids, a construction method therefor and an application thereof, the plasmids being pLHPars9 and pLLPars9, and the nucleotide sequences thereof being as shown in SEQ ID NO:1 and SEQ ID NO:2 respectively. The plasmids are sensitive to arsenite, the detection range being 0.04～50 μM, and the plasmids may be used as a biosensor for use in the detection of arsenite. In addition, arsenite inhibitory factor reporter gene plasmid pLHPars9 may further be used for detecting antimonite.

Description

A arsenite inhibitory factor reporter gene plasmid, construction method and application thereof

Technical field

The invention belongs to the technical field of genetic engineering and molecular biology, and more particularly, to a arsenite inhibitory factor reporter gene plasmid and a construction method and application thereof.

Background technique

As a naturally occurring element, arsenic is widely distributed throughout the environment. It exists in inorganic or organic form, and the inorganic form is highly toxic. The World Health Organization has set a drinking water target of 10 μg/L, but in some countries, groundwater is contaminated with arsenic concentrations above allowable levels, which poses a threat to citizens' health. Exposure to arsenic from drinking water and food for a long time can cause a variety of diseases, including cancer, cardiovascular disease, neurotoxicity and diabetes. In order to prevent further exposure to arsenic, it is necessary to quickly and cost-effective in situ analysis techniques to monitor arsenic in the water supply.

Bacterial-based assays are an emerging technology for monitoring arsenic-induced gene expression when contaminated with arsenic. The bacteria-based detection method is relatively stable and inexpensive for in situ detection of arsenic compared to conventional methods based on fixed equipment that are not suitable for in situ detection. More importantly, this measures the bioavailability of arsenic to account for the difference between exposure and dose. A key part of the bacterial-based assay is the reporter gene consisting of a promoter/operator and a reporter gene. The recent emergence of synthetic biology has facilitated the development of bacterial-based arsenic biosensors by redesigning naturally occurring bacterial operons to achieve a more sensitive response to arsenic in the environment.

The arsenic-reactive operon in E. coli can be regulated by arsenite inhibitor (ArsR) (ArsR belongs to the Smt/ArsR family), and family elements can act as transcriptional repressors, blocking RNA polymerase in the absence of arsenic compounds. Upon entry, it binds to the promoter/operator sequence of the targeted gene. After binding to arsenic, it is separated from the promoter to activate gene expression. Plasmid R773 and E. coli chromosome ArsR are homodimers, each having a Cys32-Val-Cys-Asp-Leu-Cys arsenic binding sequence located at the start of its DNA binding domain. ArsR in Thiobacillus acidophilus has no binding sequence at this position, but its cysteine residues are located at positions 95, 96 and 102. More information about the ArsR protein and its binding operons and their regulation will help to rewrite the basic molecular composition of biosensors, including inhibitors, promoters and DNA binding sites.

Various constructed reporter genes have been developed to monitor induction of gene expression. Ideally, a good reporter gene should exhibit a high sensitivity and specificity, a low endogenous background and a broad dynamic range of response. In addition, detection methods based on reporter genes should be easy to use, relatively inexpensive and safe. The autofluorescence reporter gene green fluorescent protein (GFP) is a non-invasive, ideal reporter gene. No cell lysis or analysis is required during the analysis The external substrate provides real-time detection. However, since the detection does not include the amplification function, the sensitivity is relatively low, which limits its application. Enzyme reporter genes, such as luciferase and β-galactosidase, have high sensitivity and are therefore widely used. A problem with the beta-galactosidase reporter gene is that its natural presence in bacteria leads to a high background. In the reported studies, firefly luciferase was used instead of bacterial luciferase as the reporter gene because the quantum yield of firefly luciferase was close to 90%, while bacterial luciferase was only close to 5-10%. The luciferase-based assay is fast, convenient, and has a wide linear dynamic range. In addition, its sensitivity and short half-life make it an ideal choice for measuring transient gene expression.

Summary of the invention

The object of the present invention is to overcome the deficiencies in the existing arsenic detection, and to provide arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9 and methods for constructing and using same.

In order to achieve the above object, the present invention provides a class of arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9, wherein the nucleotide sequence of pLHPars9 is shown in SEQ ID NO: 1, and the nucleotide sequence of pLLPars9 is SEQ. ID NO: 2 is shown. The arsenite inhibitory factor reporter gene plasmid of the present invention carries a common element of ArsR-luciferase and is added to E. coli and A. ferrooxidans, respectively, before the R773ArsR operon (Fig. 1). Two binding sequences of a chromosome. And the metal specificity of the two is different: pLLPars9 is specific for arsenite, and pLHPars9 is specific for both arsenite and tellurite. The only difference between pLHPars9 and pLLPars9 is the copy number of the plasmid and the corresponding ratio of ArsR to its binding promoter/operator sequence.

The method for constructing the arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9 of the present invention comprises the following steps:

(1) Using a high copy number plasmid derived from pGFPuv containing a pUC origin of replication, replacing the GFPuv gene at the high copy number plasmid XbaI and EcoRI sites with the firefly luciferase gene, and digesting with HindIII and PvuII to eliminate Lac promoter sequence;

(2) synthesizing a fragment containing a promoter region of the R 773 arsenite inhibitor operon, the nucleotide sequence is shown in SEQ ID NO: 3, and cloning the luciferase gene upstream of HindIII and XbaI to obtain pLHPars4;

(3) The fragment containing the promoter region of the R 773 arsenite inhibitor operon in step (3) is cloned together with the fragment encoding the arsenite inhibitor amino acid from positions 1 to 102 to obtain pLHPars5;

(4) synthesizing an arsenite inhibitor-binding sequence (EC-BS) containing the Escherichia coli chromosome, as shown in SEQ ID NO: 4, and containing arsenite inhibitor binding factor in Thiobacillus acidophilus Sequence of slices a segment (AF-BS), as shown in SEQ ID NO: 5, and inserted between the HindIII and PvuII sites of pLHPars5, respectively, and cloned to obtain pLHPars7 and pLHPars10;

(5) Synthesis of Escherichia coli / Thiobacillus ferrooxidans chromosome arsenite inhibitor-binding sequence (EC-BS/AF-BS), two arsenite inhibitor-binding sequences (EC) in E. coli chromosome -BS) and two arsenite inhibitory factor binding sequences (AF-BS) in the chromosome of Thiobacillus acidophilus, and cloned into pLHPars5, respectively, to obtain pLHPars9, pLHPars11 and pLHPars12;

(6) amplifying a coding region containing the EC-BS/AF-BS, the arsenite inhibitor operon and the arsenite inhibitor amino acid at positions 1 to 102 together with a fragment downstream of the luciferase gene of pLHPars9, and then Inserted between the XbaI and HindIII sites of the pACYC184 plasmid to obtain pLLPars9;

Wherein the pACYC184 plasmid was obtained from New England Biolabs (Cat# E4152S) and contained a low copy number p15A origin of replication.

Since the arsenite inhibitory factor reporter plasmids pLHPars9 and pLLPars9 of the present invention both have arsenite specificity, they can be used for the detection of arsenite. In addition, the arsenite inhibitor reporter gene plasmid pLHPars9 is also specific for tellurite and, therefore, can also be used to detect tellurite.

In order to achieve the above object, the present invention also provides an arsenite biosensor obtained by transforming Escherichia coli DH5α with the arsenite inhibitory factor reporter gene pLHPars9 and/or pLLPars9 of the present invention, the biosensor being high-copy It consists of several plasmids or low copy number plasmids and is very sensitive to arsenite.

Further, the present invention provides a tellurite biosensor which is obtained by transforming Escherichia coli DH5α with the arsenite inhibitory factor reporter gene plasmid pLHPars9 of the present invention, and is very sensitive to tellurite.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present inventors have found that the sensitivity of a biosensor based on an arsenic reporter gene depends on the maximum inhibitory property of the reporter gene in the absence of arsenic and its highest inducibility in the presence of arsenic. The present invention demonstrates that high level expression of the luciferase gene can be achieved by providing a high copy number plasmid, but this method results in low level induction, indicating that the endogenous ArsR protein is compared to the corresponding promoter/operator sequence. The amount is too low. Most promoter/operators are free of ArsR proteins for binding, resulting in no inhibition. In order to be able to modulate it, the present invention first introduces the expression of an E. coli ArsR inhibitor-luciferase fusion protein which can exert feedback inhibition on its promoter/operator. Second, two ArsR binding sequences were introduced, one from E. coli and the other from Thiobacillus acidophilus chromosome. The use of highly expressed ArsR proteins and better ArsR binding sequences enables high levels of luciferase expression through high copy number plasmids for arsenite detection.

(2) Secondly, although the expression of the luciferase gene is relatively low, the present invention can exhibit good inducibility by replacing the high copy number plasmid with a low copy number plasmid.

(3) The detection limit of the biosensor of the present invention is 0.04 μM arsenite (~5 μg/L), which is the same as the detection limit of the best biosensor reported in the literature so far. In addition, by comparing the high copy number plasmid and the low copy number plasmid based biosensor, it was found that the reaction to arsenite and the metal specificity were different, and the arsenite inhibitor reporter gene plasmid pLHPars9 was sub- Both arsenate and tellurite are specific.

DRAWINGS

The present invention and the beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

Figure 1 is a schematic diagram showing the induction of arsenic of a reporter plasmid of all the components of the present invention; wherein, after transformation of the reporter plasmid into Escherichia coli DH5α, treatment with and without 10 μM sodium arsenite for 2 hours, according to the treated cells and The treated control cells were compared, the luciferase activity was measured and the fold induction was calculated.

Figure 2 is a comparison of EMSA of the PLHPars11, pLHPars12 and pLHPars9 reporter vectors of the present invention. A indicates that E. coli DH5α cells were treated with 10 μM arsenite for 2 hours to prepare cell lysates and labeled with 2 copies of AF-BS, 2 copies of EC-BS, 1 copy of EC-BS and AF-EC biotin. The needles were mixed and subjected to EMSA analysis, and the control was mixed with the corresponding probe without arsenite-treated E. coli DH5α cell lysate. B indicates that E. coli DH5α cells were treated with 10 μM arsenite for 2 hours, and cell lysates were prepared and mixed with 1 copy of EC-BS biotin-labeled probe for EMSA analysis. The control was treated with arsenite-free E. coli DH5α. Cell lysates were mixed with 1 copy EC-BS probe.

Figure 3 is a comparison of pLHPars9 and pLLPars9 transformed cells of the present invention. Cells were transformed with pLHPars9 and pLLPars9 at concentrations ranging from 0, 0.1, 1, 10 to 100 μM for 2 and 4 hours. Cells were harvested for luciferase assay.

Figures 4 to 8 show the dynamic range and detection limit of pLHPars9 and pLLPars9 transformed cells of the present invention.

Use

0, 50, 100, 200, 400, 600 and 800 μM arsenite (Figure 4), 0, 0.2, 0.4, 0.6, 0.8 and 1.0 μM arsenite (Figures 5-6) and 0, 0.02, 0.04, 0.06, 0.08, and 0.1 μM arsenite (Figures 7-8) treated pLHPars9 and pLLPars9 transformed cells. After 1 hour of treatment, the cells were collected for luciferase analysis.

Figures 9 to 10 show the metal specificity of pLHPars9 and pLLPars9 transformed cells of the present invention. With or without 1μM K ⁺ , Na ⁺ , Mg ²⁺ , Zn ²⁺ , Ca ²⁺ , Hg ²⁺ , Sb ³⁺ , Mn ²⁺ , Ni ²⁺ , Cr ³⁺ , Co ²⁺ , Cd ²⁺ The pLHPars9 and pLLPars9 transformed cells were treated with Cr ²⁺ , Cu ²⁺ , As ⁵⁺ and As ³⁺ for 1 hour, and the cells were collected for luciferase analysis.

Detailed ways

In order to make the objects, technical solutions and beneficial technical effects of the present invention more clear, the present invention will be further described in detail below with reference to the embodiments. It is to be understood that the embodiments described in the specification are merely illustrative of the invention and are not intended to limit the invention. The parameters, proportions, and the like of the embodiments may be selected in accordance with the present invention without substantially affecting the results.

Example 1

Plasmid construction

A series of high copy number plasmids derived from pGFPuv (Clontech) containing the pUC origin of replication (ColE1). The firefly luciferase gene was used to replace the GFPuv gene at the XbaI and EcoRI sites, and digestion with HindIII and PvuII was used to eliminate the lac promoter sequence. A fragment containing the 91 bp promoter region of the R 773 ArsR operon was synthesized, and the luciferase gene upstream of HindIII and XbaI was cloned to obtain a pLHPars4 vector. A fragment containing the 91 bp promoter region was synthesized along with a fragment encoding the amino acid position 1-1-2 of ArsR, and cloned to obtain pLHPars5. Fragments containing the ArsR binding sequence in Escherichia coli chromosome (EC-BS) or the binding sequence containing Thiobacillus acidophilus (AF-BS) were separately synthesized and inserted between HindIII and PvuII sites of pLHPars5, respectively. , pLHPars7 and pLHPars10 were cloned separately. In addition, fragments of EC-BS/AF-BS, two EC-BS and two AF-BS were synthesized and cloned into pLHPars5, respectively, to obtain pLHPars9, pLHPars11 and pLHPars12, respectively. The pACYC184 plasmid obtained from New England Biolabs (Cat# E4152S) contained a low copy number p15A origin of replication. Amplification of the coding region containing the EC-BS/AF-BS, ArsR operon and ArsR amino acid positions 1 to 12 together with the downstream of the luciferase gene of pLHPars9, and then inserted between the XbaI and HindIII sites of pACYC184, pLLPars9.

The nucleotide sequence of the obtained pLHPars9 is shown in SEQ ID NO: 1, and the nucleotide sequence of pLLPars9 is shown in SEQ ID NO: 2.

In the above step, the nucleotide sequence of the fragment containing the promoter region of the R 773 arsenite inhibitor operon is as shown in SEQ ID NO: 3, and contains arsenite inhibition in the Escherichia coli chromosome (EC-BS). The factor binding sequence is set forth in SEQ ID NO: 4, and the fragment containing the binding sequence in Thiobacillus acidophilus (AF-BS) is shown in SEQ ID NO: 5, and the coding region of positions 1 to 12 of the amino acid of ArsR The nucleotide sequence is shown in SEQ ID NO: 6, and the luciferase gene sequence is shown in SEQ ID NO: 7.

2. Processing and determination

The plasmid was transformed into E. coli DH5α competent cells. Single colonies were picked and inoculated for 12-16 hours with vigorous shaking at 37 °C. Depending on the OD value, the overnight culture was diluted with the medium containing the antibiotic, the high copy number plasmid was diluted approximately 1:500, and the low copy number plasmid was diluted 1:10. The diluted cells were further cultured in a 37 ° C incubator for 3-4 hours until the O.D. value reached 0.6. 400 μL of cell fluid was taken and treated with different concentrations of sodium arsenite (Sigma). 50 μL of luciferase substrate and 20 μL of DH5α cells and 5 μL of cell lysate were mixed and measured by a luciferase assay machine (Veritas), and pLHPars9 showed high levels of luciferase gene expression.

3. Electrophoretic mobility measurement (EMSA)

Cell lysates were prepared by centrifuging 1 mL of the overnight culture at 10,000 rpm for 1 minute. The pellet was resuspended in 300 μL of lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 1 mM EDTA and 0.5% [w/v] Triton X-100) with 25 μL of freshly prepared lysozyme solution ( Incubate for 10 minutes at room temperature in 10 mM Tris-HCl at 10 mg/mL, pH 8.0). After centrifugation was completed, the supernatant was used for gel migration assay. 1-3 μg of cell lysate, 2 μL of 5 fold binding buffer and 1 μL of poly(I-C) were mixed and incubated on ice for 5 minutes. 1 μL of biotin probe was added to the mixture and incubated at 22 ° C for 30 minutes; each reaction mixture was separated from 0.5 to T 60 at 100 V 4 ° C using a 6.5% non-denaturing polyacrylamide gel. minute. After the gel was transferred to the NB membrane, 15 mL of blocking buffer was added at room temperature for 20 minutes for blocking, and the immunizing substrate was detected with streptavidin-HRP and Pierce enhanced chemiluminescent substrate. Labeled probe. Use an imager to capture images.

Experimental Example 1 Using High Copy Number Plasmids to Realize High Levels of Luciferase Gene Expression

To assess arsenite inducibility of the luciferase gene, transformed cells were treated with 10 μM sodium arsenite for 2 hours; as shown in Figure 1, no arsenite-mediated luciferase activity was observed. Induction. The E. coli chromosome ArsR acts as a trans-regulatory factor for binding to the E. coli chromosome and the plasmid R773 operon. Thus, a simple explanation for no induction is that free ArsR is predominant compared to the promoter/operator sequence that binds to ArsR because of the high copy number plasmid provided in cells expressing high levels of luciferase. The addition of arsenite removes ArsR from the promoter/operator and does not significantly alter luciferase activity compared to the unbound form because the number of promoters/operons associated with ArsR is very limited. .

Experimental Example 2 Co-expression of ArsR Reduces Basal Expression of Reporter Gene

By using feedback control of reporter gene expression, co-expression of exogenous ArsR and luciferase genes is typically utilized to reduce the basal level of luciferase activity. ArsR can be expressed by polycistronic expression or binding to a fusion protein by a reporter gene. Analysis of the deletion function by ArsR co-expressed with β-lactamase showed that the regulatory activity did not require the C-terminus of amino acids 93 to 117 of E. coli ArsR; for introduction of luciferase Feedback regulation of gene expression, a short ArsR was constructed in amino acid 1-102 of the N-terminal of luciferase to obtain the reporter gene pLHPars5 (Fig. 1). This short ArsR with amino acids 1-102 of ArsR is expressed as a fusion protein together with luciferase under the R773ArsR promoter/operator. The cells transformed with pLHPars5 were treated with 10 μM sodium arsenite for 2 hours. The luciferase assay showed 1.5-fold induction of luciferase activity (Figure 1). The inducibility of luciferase activity was not as expected.

Experimental Example 3 Adding an ArsR Binding Sequence in Front of the Ars Operon

A common approach in mammalian systems is to better determine the inducibility of the reporter gene by inserting additional copies of the cis-acting element in front of the promoter. This method has been used to reduce the underlying background of reporter gene activity during arsenite biosensor construction by inserting an additional copy of the E. coli ArsR binding sequence between the ArsR gene and the reporter gene. It showed a slightly lower background compared to the control reporter gene without the addition of additional ArsR binding sequences. In the present invention, an E. coli ArsR binding sequence (EC-BS) or a Thiobacillus acidophilus ArsR binding sequence (AF-BS) is constructed in front of the R773ArsR promoter/operator of pLHPars5 to obtain the reporter gene vector pLHPars7 and pLHPars10 (Figure 1). Treatment of DH5α-transformed cells with 10 μM arsenite for 2 hours showed a 2-fold induction of luciferase activity (Fig. 1). There was no significant improvement in pLHPars7 and pLHPars10 in the reaction with arsenite compared to pLHPars5 without the addition of additional ArsR binding sequences. Next, 2 copies of EC-BS or AF-BS, and 1 copy of each of EC-BS and AF-BS were added in front of the promoter/operator of pLHPars5, respectively, and pLHPars11, pLHPars12, and pLHPars9 were obtained (Fig. 1). The reporter gene vector pLHPars11 with 2 copies of EC-BS and pLHPars12 with 2 copies of AF-BS showed no effect in arsenite induction (Fig. 1). Notably, the reporter gene pLHPars9 with 1 copy of EC-BS and 1 copy of AF-BS exhibited a 5-fold luciferase induction fold after 2 hours of treatment with 10 μM arsenite (Figure 1). , better than 2 copies of EC-BS or AF-BS. These results indicate that the two binding sequences together can complement each other such that they are superior to the addition of 2 copies of EC-BS or AF-BS alone in E. coli ArsR binding.

Experimental Example 4 Differences in ArsR in its binding sequence

In order to directly test the interaction with E. coli ArsR, whether EC-BS and AF-BS alone are different from 2 copies of EC-BS or AF-BS, EMSA was used to prepare cell lysate from Escherichia coli DH5α-pLHPars5. And treated with 10 μM arsenite for 2 hours. EMSA was performed using cell lysates instead of purified ArsR proteins to determine natural interactions. Biotin-labeled probes were chemically synthesized at the 5' end according to the sequences of EC-BS and AF-BS. A probe with 2 copies of EC-BS, 2 copies of AF-BS, and 1 copy of each of AF-BS and EC-BS was synthesized for EMSA analysis. Figure 2A shows the result, AF-BS-EC-BS The combination was stronger than 2 copies of EC-BS and 2 copies of AF-BS, consistent with the induction data. This indicates that ArsR can be spatially combined with 1 copy of AF-BS and EC-BS, better than alone with EC-BS or AF-BS. A better ArsR binding sequence was found by an optimization scheme and several key positions in the ArsR binding sequence were determined. One copy of EC-BS and AF-BS contains nucleotides at the junction of EC-BS and AF-BS, which contributes to better interaction with E. coli ArsR.

In addition, using a probe with 2 copies of the binding sequence, there will be two distinct shift bands in the EMSA results, indicating that there are two possibilities, the ArsR dimer and the tetramer coexist or both forms DNA probe; one with one ArsR dimer and the other with two ArsR dimers. In the former possibility, dimers and tetramers coexist, while in another possibility only dimers are present. If a dimer and a tetramer coexist, both should be detected even if one DNA binding sequence is used as a probe. As shown in Figure 2B, only one shift band was observed, corresponding to the lower band in Figure 2A, indicating that only ArsR dimers were present rather than coexisting with the tetramer. Thus, the two shift bands with probes consist of one dimer and two dimers, respectively, of ArsR.

The addition of arsenite induces the dissociation of ArsR from the binding sequence, consistent with the above conclusions, and the inhibitory factor needs to be separated from the promoter. Unlike the MerR family of inhibitors, there is no need for dissociation, but metal binding induces a conformation that shifts from a poor promoter to a stronger promoter.

Experimental Example 5 Comparison of Biosensors with High Copy Number Plasmids and Low Copy Number Plasmids

To examine whether the inducibility was different when the promoter/operator copy number was low, a low copy number p15A origin of replication from plasmid pACYC184 was introduced, instead of the high copy number pUC origin of replication, pLLPars9 was obtained. Treatment of pLLPars9 transformed cells with 10 [mu]M arsenite for 2 hours showed >10-fold induction compared to untreated control cells, superior to pLHPars9 (Figure 1). To more specifically compare pLHPars9 and pLLPars9, transformed cells were treated with a wide range of concentrations of arsenite and treated with 0, 0.1, 1, 10 to 100 μM arsenite for 2 and 4 hours. As expected, the basal level of luciferase in pLLPars9 transformed cells was much lower than that of pLHPars9, while the maximum level was also significantly lower (Fig. 3). Using a high copy number plasmid, inducibility can occur with a sharp change of 0.1 to 1 μM in 2 hours, while arsenite changes more slowly within this range within 4 hours. A decrease in luciferase activity of pLHPars9 was observed within 4 hours using 100 μM arsenite, but was not observed at 2 hours. The induction pattern with a low copy number plasmid differs from the arsenite treated high copy number plasmid. In addition, no decrease in luciferase activity was observed under conditions with a low copy number plasmid. These results indicate that the arsenite inducibility of luciferase differs between high copy number and low copy number plasmids.

Experimental Example 6 Dynamic range and detection limit of DH5α cells transformed with pLHPars9 and pLLPars9

The application of biosensors in this field is expected to be completed in a short time. In some studies, the shortest time to treat with arsenic compounds was 1 hour. Cells transformed with pLHPars9 or pLLPars9 were treated with arsenite at various concentration ranges (0, 50, 100, 200, 400, 600 to 800 μM) for 1 hour. Luciferase assays showed that the pLHPars9 and pLLPars9 biosensors peaked at 50 μM and 100 μM arsenite concentrations, respectively, after which activity began to decline (Figure 4). In addition, treatment of both cells with 0, 0.2, 0.4, 0.8 to 1.0 μM arsenite for 1 hour showed a linear response (Figs. 5 to 6). To investigate the detection limit, cells were treated with 0, 0.02, 0.04, 0.06, 0.08 and 0.16 μM arsenite for 1 hour. The pLHPars9 and pLLPars9 biosensors can both display 0.04 μM arsenite if the detection limit for arsenite concentration that causes significant induction is defined as background plus 2 standard deviations (SD).

The detection limit (Figures 7-8) is below the 10 μg/L World Health Organization (WHO) guidelines. Therefore, this indicates that both can be used as arsenite biosensors.

Experimental Example 7 Differences in Selective Reactions of Metals Between pLHPars9 and pLLPars9 Biosensors

The specificity of the two biosensors was tested and compared to 17 different metals. Generally, as shown in Figures 9-10, the biosensor pLLPars9 can show more specificity than pLHPars9. Among the metals tested, pLLPars9 was only induced by arsenite, although it was slightly inhibited by Hg(II), but no significant induction was observed when using arsenate and other metals. The biosensor pLHPars9 showed a large difference from pLLPars9. In addition to the reaction with arsenite As (III), it also reacts with the tellurite Sb (III) and slightly reacts with the arsenate As (V). The difference between pLLPars9 and pLHPars9 is the copy number of its promoter/operator and its corresponding ratio to the ArsR protein. Thus, the metal specificity of ArsR can be modulated by the concentration of the ArsR protein, the ArsR binding promoter/operator, or the ratio of ArsR to its binding promoter/operator.

The above embodiments may be modified and modified as appropriate by those skilled in the art in light of the above disclosure. Therefore, the invention is not limited to the specific embodiments disclosed and described herein, and the modifications and variations of the invention are intended to fall within the scope of the appended claims. In addition, although specific terms are used in the specification, these terms are merely for convenience of description and do not limit the invention.

Claims

A class of arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9, characterized in that the nucleotide sequence of pLHPars9 is shown in SEQ ID NO: 1, and the nucleotide sequence of pLLPars9 is shown in SEQ ID NO: 2.
The method for constructing the arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9 according to claim 1, comprising the steps of:

(1) Using a high copy number plasmid derived from pGFPuv containing a pUC origin of replication, replacing the GFPuv gene at the high copy number plasmid XbaI and EcoRI sites with the firefly luciferase gene, and digesting with HindIII and PvuII to eliminate Lac promoter sequence;

(2) synthesizing a fragment containing a promoter region of the R 773 arsenite inhibitor operon, the nucleotide sequence is shown in SEQ ID NO: 3, and cloning the luciferase gene upstream of HindIII and XbaI to obtain pLHPars4;

(3) The fragment containing the promoter region of the R 773 arsenite inhibitor operon in step (3) is cloned together with the fragment encoding the arsenite inhibitor amino acid from positions 1 to 102 to obtain pLHPars5;

(4) synthesizing a fragment containing an arsenite inhibitor-inducing factor in the chromosome of Escherichia coli, as shown in SEQ ID NO: 4, and a fragment containing an arsenite inhibitor-binding sequence in the chromosome of Thiobacillus acidophilus As shown in SEQ ID NO: 5, and inserted into the two sites of HindIII and PvuII of pLHPars5, respectively, and cloned to obtain pLHPars7 and pLHPars10;

(5) Synthetic Escherichia coli / Thiobacillus ferrooxidans chromosome arsenite inhibitor binding sequence, two E. coli chromosome arsenite inhibitor binding sequences and two E. acidophilus chromosomes The arsenate inhibitory factor binding sequence was cloned into pLHPars5 to obtain pLHPars9, pLHPars11 and pLHPars12, respectively;

(6) Encoding the coding of the arsenite inhibitor-inhibiting sequence, the arsenite inhibitor operon and the arsenite inhibitor amino acid in the chromosome of Escherichia coli/E. coli The region, together with a fragment downstream of the luciferase gene of pLHPars9, was inserted between the XbaI and HindIII sites of the pACYC184 plasmid to obtain pLLPars9;

Wherein the pACYC184 plasmid contains a low copy number p15A origin of replication.
Use of the arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9 of claim 1 in the detection of arsenite.
Use of the arsenite inhibitory factor reporter gene plasmids pLHPars9 and pLLPars9 according to claim 3, characterized in that the arsenite inhibitory factor reporter gene plasmid pLHPars9 is also used for the detection of tellurite.
An arsenite biosensor obtained by transforming E. coli DH5α with the arsenite inhibitory factor reporter gene plasmid pLHPars9 and/or pLLPars9 according to claim 1.
A tellurite biosensor obtained by transforming Escherichia coli DH5α with the arsenite inhibitory factor reporter gene plasmid pLHPars9 according to claim 1.