CN111979166B

CN111979166B - Engineering bacterium for specifically removing arsenic as well as construction method and application thereof

Info

Publication number: CN111979166B
Application number: CN202010820345.7A
Authority: CN
Inventors: 李琴; 伍一军; 杨文耀; 王菲菲; 车飞; 张艳平; 王海燕; 王宗爽
Original assignee: Institute of Zoology of CAS; Chinese Research Academy of Environmental Sciences
Current assignee: Institute of Zoology of CAS; Chinese Research Academy of Environmental Sciences
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2022-11-18
Anticipated expiration: 2040-08-14
Also published as: CN116064627A; CN111979166A

Abstract

The invention relates to the technical field of arsenic removal by a biological method, in particular to an engineering bacterium for specifically removing arsenic, a construction method and application thereof. The engineering bacteria provided by the invention comprise an arsenic binding protein expression cassette, wherein the expression cassette comprises an arsenic induction promoter, a bacteria surface display carrier protein gene, a fluorescent protein gene and an arsenic binding protein gene. The engineering bacteria can efficiently express the arsenic binding protein with arsenic binding activity on the surface of the bacteria under the induction of arsenic, does not need to add an inducer additionally, can specifically bind the arsenic, and can monitor the expression of the arsenic binding protein according to a fluorescent signal. The engineering bacteria for specifically removing arsenic provided by the invention provides a new method for treating arsenic pollutants in the environment, and meanwhile, the engineering bacteria provide a new thought, means and resource for constructing the engineering bacteria with the function of removing specific metals by using the metal specific induction promoter and the corresponding metal specific regulatory protein, and have important significance in removing non-metal and metal pollutants in the environment.

Description

Engineering bacterium for specifically removing arsenic as well as construction method and application thereof

Technical Field

The invention relates to the technical field of arsenic removal by a biological method, in particular to an engineering bacterium for specifically removing arsenic, a construction method and application thereof.

Background

At present, the main methods for remedying environmental pollution include a physicochemical method for removing pollutants from the environment by a physicochemical means or converting them into stable harmless substances, and a biological method for removing pollutants from the environment by the characteristics of organisms or certain components in the organisms.

The removal of metal ions can be achieved by utilizing the adsorption capacity of the bacteria expressing the metal binding protein to the metal ions. At present, the research on metal binding protein genetic engineering bacteria is limited to the construction of non-specific metal binding genetic engineering bacteria (having adsorption effect on various metal ions), or an inducer is often required to be added when the metal binding protein is expressed, or the binding and adsorption capacity on metals is limited. The simultaneous adsorption of various heavy metal ions is not beneficial to the recycling of the adsorbed heavy metals; the addition of the inducer not only increases the economic cost, but also some inducers (such as IPTG) have certain toxicity; the limited binding capacity of metals results in less efficient metal removal or recovery.

Arsenic and its compounds are common environmental pollutants, and arsenic and arsenide pollutants in the environment can enter human bodies through water, atmosphere, food and other ways, thus causing harm. The discovery of the promoter with specific response to the environmental pollutants and the protein capable of being specifically combined with the environmental pollutants provides possibility for constructing the engineering bacteria with specific adsorption effect on the pollutants. On the basis, the construction of the engineering bacteria which can play a role in specifically adsorbing arsenic without specifically adding an inducer has important significance.

Disclosure of Invention

The invention aims to provide an engineering bacterium for specifically removing arsenic, and the invention also aims to provide a construction method and application of the strain.

In order to realize the purpose, the invention utilizes the gene recombination technology to clone the arsenic binding protein gene and the bacterial surface display carrier protein gene to the downstream of an arsenic-induced promoter, and constructs an expression cassette which can express the arsenic binding protein on the surface by arsenic regulation; the expression cassette is transferred into host bacteria to construct engineering bacteria with specific adsorption effect on arsenic. When arsenic exists in the environment, the engineering bacteria can express arsenic binding protein and specifically adsorb and remove the arsenic without adding an additional inducer. On the basis of the engineering bacteria, a fluorescent protein gene is further introduced into the arsenic binding protein expression cassette, so that the expression condition of the arsenic binding protein can be monitored according to a fluorescent signal.

The arsenic binding protein expression cassette comprises a fusion protein of a bacterial surface display carrier protein, a fluorescent protein and an arsenic binding protein, wherein each protein in the fusion protein needs to have a normal protein structure to play a normal function, and unpredictable mutual influences exist between the expression and folding of each protein in the fusion protein, so that the expression quantity and correct folding of one or more proteins in the fusion protein are influenced. When constructing a fusion protein and an expression cassette containing a bacterial surface display carrier protein, a fluorescent protein and an arsenic binding protein, the invention finds that different promoters, arsenic binding proteins and bacterial surface display carrier proteins are selected to be combined to obtain the expression cassette with larger difference in surface expression effect of the arsenic binding proteins.

Specifically, the invention provides the following technical scheme:

the invention firstly provides an engineering bacterium, which comprises a metal or nonmetal binding protein expression box, wherein the expression box comprises a metal-induced promoter or nonmetal-induced promoter, a bacterial surface display carrier protein gene, a metal binding protein gene or a nonmetal binding protein gene.

The metal is cadmium, zinc, lead, copper and nickel, and the nonmetal is arsenic.

Further, the invention provides an engineering bacterium for specifically removing arsenic, which comprises an arsenic binding protein expression cassette, wherein the expression cassette comprises an arsenic induction promoter, a bacterium surface display carrier protein gene and an arsenic binding protein gene.

The arsenic binding protein is preferably derived from Escherichia coli genome or Escherichia coli R factor R773 plasmid, pseudomonas fluorescens MSP3 genome, and Pseudomonas aeruginosa genome.

Preferably, the examples of the present application provide the arsenic-inducible promoter described above, which are Pars1 (comprising an operator, i.e., a regulatory region sequence, a promoter sequence, and a regulatory gene, i.e., a gene sequence encoding a transcriptional repressor that specifically binds arsenic, and having a total of 492 bp) in the examples, and Pars2 (comprising an operator, i.e., a regulatory region sequence, a promoter sequence, and a regulatory gene, i.e., a gene sequence encoding a transcriptional repressor that specifically binds arsenic, and having a total of 451 bp), having a nucleotide sequence shown in SEQ No. 2.

As an embodiment of the invention, the nucleotide sequence of the arsenic-inducible promoter is shown in SEQ ID No.1, and the nucleotide sequence of the arsenic-binding protein gene is shown in SEQ ID No. 3.

As another embodiment of the invention, the nucleotide sequence of the promoter is shown as SEQ ID NO.1, and the nucleotide sequence of the arsenic binding protein gene is shown as SEQ ID NO. 4.

As a preferred embodiment of the invention, the nucleotide sequence of the promoter is shown as SEQ ID NO.2, and the nucleotide sequence of the arsenic binding protein gene is shown as SEQ ID NO. 3. The engineering bacteria carrying the expression cassette have better arsenic binding protein surface expression and arsenic binding activity, and the arsenic adsorption activity is better under the condition of high arsenic concentration (for example, the arsenic concentration is more than or equal to 40 mu mol/L or the arsenic concentration is more than or equal to 160 mu mol/L).

The bacterial surface display carrier protein is one selected from the group consisting of an ice nucleoprotein, lpp-OmpA and OmpC. Preferably the Ice Nucleation Protein (INP) N-terminal domain.

To facilitate monitoring of arsenic binding protein expression, the expression cassettes of the invention also comprise a fluorescent protein gene.

Preferably, the expression cassette comprises an arsenic-inducible promoter, a bacterial surface display carrier protein gene, a fluorescent protein gene and an arsenic-binding protein gene in sequence from 5 'to 3'.

The expression cassette of the invention preferably has a sequence as shown in any one of SEQ ID NO. 5-7.

Wherein, the 1 st to 492 st of the sequence shown as SEQ ID NO.5 are arsenic induced promoter system sequence, the 599 th to 1171 th are ice nucleoprotein N-terminal domain sequence, the 1178 th to 1885 th are fluorescent protein gene sequence, and the 1892 th to 2245 th are arsenic binding protein sequence. As shown in SEQ ID NO.6, the 1 st to 451 th sites of the sequence are arsenic-inducing promoter system sequence, the 560 th to 1132 th sites are ice nucleoprotein N-terminal domain sequence, the 1139 th to 1846 th sites are fluorescent protein gene sequence, and the 1853 rd to 2206 th sites are arsenic-binding protein sequence. As shown in SEQ ID NO.7, the 1 st to 492 th sites of the sequence are arsenic-induced promoter sequences, the 599 th to 1171 th sites are ice-nucleating protein N-terminal domain sequences, the 1178 th to 1885 th sites are fluorescent protein gene sequences, and the 1892 th to 2245 th sites are arsenic-binding protein sequences.

The expression cassette of the invention may further comprise a transcription terminator located downstream of the arsenic-binding protein gene. The transcription terminator is preferably rrnBT1T2.

The expression cassette can be inserted into the genome of the engineering bacteria or inserted into a plasmid vector to independently replicate and inherit.

Aiming at the expression cassette, the invention screens the adapted host bacteria which can realize the high-efficiency and correct expression of the arsenic binding protein on the bacterial surface.

Preferably, the engineering bacterium provided by the invention is Escherichia coli (Escherichia coli) containing a vector carrying the expression cassette. The Escherichia coli is preferably Escherichia coli BL21 strain.

The plasmid vector into which the above-described expression cassette is inserted may be a pUC series vector, a pBR322 series vector, or a pACYC series vector.

The invention also provides a construction method of the engineering bacteria for specifically removing arsenic, which comprises the following steps: and (3) introducing the expression cassette or a vector carrying the expression cassette into host bacteria.

The invention provides a preparation containing the engineering bacteria.

The preparation can be a liquid preparation or a solid preparation, and is prepared by adding the engineering bacteria into auxiliary materials allowed in the field of microbial preparations.

The invention also provides application of the engineering bacteria or the preparation in removing or recovering metal or nonmetal elements, or application in treating metal or nonmetal element pollution in the environment. Preferably, the invention provides the use of the engineered bacteria or the preparation for arsenic removal or recovery, or for the treatment of arsenic contamination in an environment. The invention has the beneficial effects that: the invention constructs an arsenic-binding protein expression cassette, a vector and an engineering bacterium which are regulated and controlled by arsenic, can express the arsenic-binding protein on the surface of the bacterium and have fluorescence characteristics by placing a fusion protein gene of a bacterium surface display carrier protein-fluorescent protein-arsenic binding protein at the downstream of an arsenic induction promoter. The engineering bacterium can efficiently express the arsenic binding protein with arsenic binding activity on the surface without adding an inducer additionally, has specific arsenic binding characteristic, and can monitor the expression of the arsenic binding protein according to a fluorescent signal. The engineering bacteria for specifically removing arsenic provided by the invention provide a new method and a new idea for treating arsenic pollutants in the environment, and have a better application prospect. The engineering bacteria for specifically removing arsenic provided by the invention provides a new method for treating arsenic pollutants in the environment, and meanwhile, the engineering bacteria provided by the invention provide a new thought, means and resource for constructing the engineering bacteria with the function of removing specific metal by using the metal specific induction promoter and the corresponding metal specific regulatory protein, and have important significance in the aspect of removing non-metal or metal pollution in the environment.

Drawings

FIG. 1 shows the results of the growth and fluorescence intensity detection of induced bacteria and non-induced bacteria in the method for constructing an As-GP vector in example 1 of the present invention.

FIG. 2 shows the results of the growth and fluorescence intensity detection of induced bacteria and non-induced bacteria in the method for constructing an As2-GP vector in example 1 of the present invention.

FIG. 3 is a schematic view showing the structure of an As2-ICAR (A21) carrier in example 2 of the present invention.

FIG. 4 is a schematic view showing the structure of As1-ICAR (A11) carrier in example 3 of the present invention.

FIG. 5 is a schematic view showing the structure of As1-ICAR2 (A12) vector in example 4 of the present invention.

FIG. 6 is a graph showing the growth of various strains containing As1-ICAR (A11) vector in example 5 of the present invention.

FIG. 7 shows the expression of fluorescent proteins of different strains containing As1-ICAR (A11) vector in example 5 of the present invention.

FIG. 8 is a graph showing the growth of various strains containing As2-ICAR (A21) vector in example 5 of the present invention.

FIG. 9 shows the expression of fluorescent proteins of different strains containing As2-ICAR (A21) vector in example 5 of the present invention.

FIG. 10 is a graph showing the growth of various strains containing As1-ICAR2 (A12) vector in example 5 of the present invention.

FIG. 11 shows the expression of fluorescent proteins of different strains containing As1-ICAR2 (A12) vector in example 5 of the present invention.

FIG. 12 shows the results of comparing the arsenic removal effects of different engineering bacteria in example 6 of the present invention.

In FIGS. 4 to 10 above, A21-Top10 represents the E.coli Top10 strain containing the As2-ICAR (A21) vector; A21-BL21 represents an E.coli BL21 strain containing an As2-ICAR (A21) vector; A21-DH 5. Alpha. Represents E.coli DH 5. Alpha. Strain containing As2-ICAR (A21) vector; the strains represented by other notations can be determined by reference to this nomenclature.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The experimental procedures used in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 construction of the basic vector

1. Construction of pUC-As vector

(1) Sequence design: a DNA sequence with the length of 662bp is designed, wherein an arsenic induction promoter system (comprising an operator gene, namely a regulatory region sequence, a promoter sequence, and a regulatory gene, namely a transcription repressor gene sequence capable of being specifically combined with arsenic) is contained in the DNA sequence (refer to GenBank Accession No. X16045.1, the arsenic induction promoter from the source is named as 'Pars 1' in the invention, the nucleotide sequence of the arsenic induction promoter is shown as SEQ ID NO.1, the transcription repressor specifically combined with arsenic is named as 'ArsR 1', the nucleotide sequence of the transcription repressor is shown as SEQ ID NO. 3), a second regulatory region sequence, a nucleoprotein body combining site and a polyclonal enzyme cutting site.

(2) Construction of pUC-As vector: the 662bp DNA fragment (Biotechnology engineering (Shanghai) Co., ltd.) was synthesized by a total gene synthesis method and cloned between Sac I and Hind III restriction sites of a pUC57 plasmid (Biotechnology engineering (Shanghai) Co., ltd.), and finally a pUC-As vector was constructed. The accuracy of the synthetic gene sequence and the accuracy of the gram Long Meiqie site were all verified by sequencing.

2. Construction of As-BTT vector

The purpose of this step was to add a transcription terminator to the pUC-As vector.

(1) Construction of As-BTT vector: a472 bp rrnBT1T2 fragment (which contains the transcription terminator required by the present invention) was PCR amplified using the pBV220 plasmid (commercially available vector) as a template. The amplification system used is 25. Mu.L of Premix ExTaq reagent (TaKaRa), the sequence of the upstream primer is 5'-AACTGCAGAGCTTCTGTTTTGGCGGATG (PstI), and the sequence of the downstream primer is 5' -ATGCATGCCATGAGCGGATACATATTTGAATG (SphI). Purifying and recovering rrnBT1T2 fragment by agarose gel electrophoresis, and TA cloning the fragment (A: (A))

19-T vector, taKaRa), double digested with PstI and SphI to obtain rrnBT1T2 fragment from T vector, which was ligated to pUC-As vector fragment constructed in step 1 after double digested with PstI and SphI (T4 DNA ligase, taKaRa, operating As per instructions).

(2) Transformation and identification: e.coli DH 5. Alpha. Was transformed with the ligation product of step (1), spread on LB agar plate containing ampicillin (50. Mu.g/mL, the same applies below), and cultured overnight at 37 ℃. Single colonies were picked up in liquid LB containing ampicillin (volume 5 mL) and cultured overnight at 37 ℃ at 200-250 r/min. And (4) extracting plasmids from overnight culture liquid, and sequencing to verify the accuracy of the sequence.

3. Construction of As-GP vectors

In order to test the arsenic induction expression function of the constructed As-BTT vector, a fluorescent protein reporter gene is inserted into the As-BTT vector, the expression of the fluorescent protein is observed by adding an arsenic inducer, if the fluorescent protein is normally expressed, namely thalli present fluorescence characteristics, the As-BTT vector has arsenic induction expression characteristics, and an arsenic induction expression system constructed based on Pars1 is correct.

(1) Preparation of a linearized vector: the As-BTT vector obtained in step 2 was constructed by double digestion with NcoI and PstI, and the desired band was recovered and purified by gel electrophoresis.

(2) Preparation of a DNA fragment of interest: the sfGFP fragment, 717bp, was amplified using the sfGFP plasmid (Shanghai Linyuan Biotech Co., ltd.) as a template. Pfu DNA polymerase (Suzhou Hongsn Biotechnology Ltd.) was used, the upstream primer sequence was 5'-TGTTGTGGATCCAAGAAGGAGATATACCATGGCAATGCGTAAAGGCGAAG-3' (NcoI) and the downstream primer sequence was 5'-TCTCTCATCCGCCAAAACAGAAGCTCTGCAGTCATTTGTACAGTTCATC-3' (PstI).

(3) Construction of As-GP vectors: a recombinant reaction system was prepared on ice, the total volume was 20. Mu.L, 10. Mu.L (volume ratio 1:1) of the recombinant reaction system containing the target DNA fragment and the linearized vector and 10. Mu.L of 2 Xrecombinase reaction solution (Hongxin Biotechnology Ltd., suzhou) were reacted at 50 ℃ for 60 minutes, and then cooled on ice.

(4) Transformation and identification: 10. Mu.L of the recombinant reaction solution was used to transform E.coli DH 5. Alpha. Competent cells, which were plated on LB agar plate containing ampicillin (50. Mu.g/ml, the same applies below), and cultured overnight at 37 ℃. Single colonies were picked up in liquid LB containing ampicillin (final ampicillin concentration 50. Mu.g/mL, volume 5 mL) and cultured overnight at 37 ℃ at 200-250 r/min. And (4) extracting plasmids from overnight culture liquid, and sequencing to verify the accuracy of the sequence.

(5) Expression of fluorescent protein: a monoclonal colony of E.coli DH5 alpha containing an As-GP carrier is selected and inoculated in an LB liquid culture medium containing ampicillin, when the OD600 value is about 0.5-0.8 during culture at 37 ℃, the bacterial liquid is divided into 2 parts, one part of bacterial liquid (induced bacteria) is added with an arsenic inducer solution with the final concentration of arsenic of 30 mu mol/L, and the other part of bacterial liquid (non-induced bacteria) is added with sterilized deionized ultrapure water to replace the arsenic inducer solution. The time for adding the arsenic inducer is 0h, the OD600 and the fluorescence intensity value of the bacterial liquid are respectively detected when the inducer is added for culturing for 4h, and the growth and fluorescence intensity detection results of each strain are respectively shown in figure 1. As can be seen from the results in FIG. 1, the fluorescence intensity value of the induced bacteria is much higher than that of the non-induced bacteria, which indicates that the As-GP vector has the arsenic-induced expression characteristic, and the arsenic-induced promoter can normally function.

4. Construction of As2-GP vector

The escherichia coli genome contains an arsenic-induced promoter different from the sequence (the invention names the arsenic-induced promoter sequence derived from the E.coli DH5 alpha genome As 'Pars 2', the nucleotide sequence of the arsenic-induced promoter sequence is shown As SEQ ID NO.2, the transcription repressor protein specifically bound with arsenic is named As 'ArsR 2', the nucleotide sequence of the transcription repressor protein is shown As SEQ ID NO. 4), a Pars2 fragment comprising an operator gene, namely a regulatory region sequence, a promoter sequence and a regulatory gene, namely a gene sequence encoding the transcription repressor protein capable of specifically binding with arsenic is cloned from the E.coli DH5 alpha genome, the arsenic-induced promoter in the As-GP vector is replaced by the Pars2 fragment to form an As2-GP vector, the expression of the fluorescent protein is observed by adding an arsenic inducer, if the fluorescent protein is normally expressed, namely, thallus presents the fluorescent characteristic, the Pars2 fragment has the arsenic-induced expression function, and the Pars2 fragment does not contain a second regulatory region sequence.

(1) Extraction of coli DH5 α genome: 1.5ml of overnight-cultured E.coli DH5 alpha bacterial liquid is taken, and E.coli DH5 alpha genome is extracted by adopting a bacterial genome DNA extraction kit (centrifugal column method) (Beijing Pubo Xin Biotechnology, inc.), and the specific operation is shown in an instruction book of the Beijing Pubo Xin Biotechnology, inc.

(2) As2-GP vector construction: PCR amplification of Pars2 fragment (including operator gene, i.e. regulatory region sequence, promoter sequence, regulatory gene, i.e. code capable of specific binding with arsenic) by using extracted DH5 alpha genome as templateThe arsenic-inducible promoter sequence derived from the e.coli DH5 α genome is named "Pars2" and the transcription repressor protein to which arsenic specifically binds is named "ArsR 2"): the PCR reaction premixing kit Premix Taq from TaKaRa company is adopted ^TM (Ex Taq ^TM Version 2.0), the upstream primer sequence is: 5'-AGGAATTCCCGCGGTTACCTTCCTCT GCACTTACAC-3' (EcoRI) and the downstream primer sequence 5'-CGACGGATCCTTA ACTGCAAATGTTCTTACTGTCCCC-3' (BamHI). Agarose gel electrophoresis to recover and purify the PCR target fragment, TA cloning the fragment (

19-T vector, taKaRa corporation), double-digested with EcoRI and BamHI from the T vector to obtain Pars2 fragment, which was ligated to EcoRI and BamHI sites in As-GP constructed in step 3 (the As-GP vector was digested with EcoRI and BamHI, and large fragment was recovered for ligation), and ligase was performed using T4 DNA ligase (TaKaRa corporation) according to the instructions.

(3) Transformation and identification: coli TOP10 competent cells were transformed with the ligation mixture, plated on LB agar plates containing ampicillin (50. Mu.g/ml, the same applies below), and cultured overnight at 37 ℃. Single colonies were picked up in liquid LB containing ampicillin (final ampicillin concentration 50. Mu.g/mL, volume 5 mL) and cultured overnight at 37 ℃ at 200-250 r/min. And (3) extracting plasmids from overnight culture liquid, and verifying the accuracy of the sequence by sequencing.

(4) Expression of fluorescent protein: selecting a monoclonal colony, inoculating the colony in an LB liquid culture medium containing ampicillin, dividing the bacterial liquid into 2 parts when the OD600 value is about 0.5-0.8 at 37 ℃ for culture, adding an arsenic inducer solution with the final concentration of 30 mu mol/L of arsenic into one part of bacterial liquid (induced bacteria), and adding sterilized deionized ultrapure water into the other part of bacterial liquid (non-induced bacteria) to replace the arsenic inducer solution. The time for adding the arsenic inducer is 0h, the OD600 and the fluorescence intensity value of the bacterial liquid are respectively detected when the inducer is added for culturing for 4h, and the growth and fluorescence intensity detection results of each strain are respectively shown in figure 2. As can be seen from the results of FIG. 2, the fluorescence intensity value of the induced bacteria is much higher than that of the non-induced bacteria, which indicates that the As2-GP vector has the arsenic-induced expression characteristic, and the arsenic-induced promoter can normally play a role.

EXAMPLE 2 construction of As2-ICAR (A21) As-binding protein expression vector

This example constructed an As2-ICAR (A21) vector in order to obtain an expression vector in which expression of the arsenic-specific binding protein ArsR1 was controlled by a Pars2 arsenic-inducible promoter, by replacing the ribosome binding site sequence-sfGFP sequence in the As2-GP vector with a fragment of the designed and synthesized ICAR sequence. The designed and synthesized ICAR sequence comprises a regulatory region sequence of an arsenic-induced promoter Pars2, a nucleoprotein body binding site sequence, an N-terminal structural domain sequence of an iceryprotein inaK gene, a red fluorescent protein mCherry sequence and an arsenic-binding protein ArsR1 sequence, wherein the regulatory region sequence of the arsenic-induced promoter Pars2 added in the ICAR sequence is a second regulatory region sequence of Pars2 in an As2-ICAR vector.

The As2-GP vector prepared in the example 1 comprises an arsenic-inducible promoter system and a fluorescent protein sfGFP, the fluorescent protein sfGFP sequence is cut off in the A21 construction process, an ICAR sequence fragment is inserted at the same time, a regulatory region sequence of an arsenic-inducible promoter (Pars 2), namely a repression site, is included at the front end of the ICAR sequence fragment, and the background expression of the arsenic-inducible promoter can be better controlled by increasing the regulatory region sequence.

An ICAR sequence fragment (Suzhou Honghen Biotechnology Co., ltd.) with length of 1761bp, which is designed by whole gene synthesis, comprises a regulatory region sequence of an arsenic induction promoter Pars2, a nucleoprotein body binding site sequence, an N-end structural domain sequence of an ice nucleoprotein inaK gene, a red fluorescent protein mCherry sequence and an arsenic binding protein ArsR1 sequence, and then the ICAR fragment is cloned between BamHI and PstI enzyme cutting sites of an As2-GP vector to construct an As2-ICAR vector (figure 3).

The accuracy of the synthetic gene sequence and the accuracy of the gram Long Meiqie locus were all verified by sequencing. The nucleotide sequence of the arsenic-induced promoter of the vector A21 (As 2-ICAR vector) is shown As SEQ ID NO.2, and the nucleotide sequence of the arsenic-binding protein gene is shown As SEQ ID NO. 3; the nucleotide sequence of the expression cassette is shown as SEQ ID NO.6, and comprises an arsenic-induced promoter (1-451 bp), an ice nucleoprotein N-terminal domain sequence (560-1132 bp), a fluorescent protein gene (1139-1846 bp), an arsenic binding protein gene (1853-2206 bp) and rrBT1T2 (2213-2684 bp).

EXAMPLE 3 construction of As-binding protein expression vector As1-ICAR (A11)

The As1-ICAR (A11) vector was constructed in this example in order to obtain an expression vector in which the expression of the arsenic-specific binding protein ArsR1 was controlled by the Pars1 arsenic-inducible promoter, by replacing the arsenic-inducible promoter expression system in the As2-ICAR (A21) vector with a Pars1 fragment containing the entire arsenic-inducible promoter expression system obtained from the As1-GP vector. The Pars1 fragment contains a corresponding operator gene, namely a regulatory region sequence, a promoter sequence, a regulatory gene, namely a gene sequence encoding a transcription repressor protein capable of being specifically combined with arsenic, and a second regulatory region sequence.

A Pars1 fragment (602 bp) containing a complete arsenic-induced initiation expression system is obtained by double digestion of the As1-GP vector with SacII and NcoI, a large fragment (4786 bp) containing an ICAR fragment is obtained by double digestion of the As2-ICAR vector with SacII and NcoI, and the Pars1 fragment and the large fragment (4786 bp) containing the ICAR fragment are connected to form the As1-ICAR vector (FIG. 4). The accuracy of the sequence was verified by sequencing. The nucleotide sequence of the arsenic-induced promoter of the vector A11 (As 1-ICAR) is shown As SEQ ID NO.1, and the nucleotide sequence of the arsenic-binding protein gene is shown As SEQ ID NO. 3; the nucleotide sequence of the expression cassette is shown as SEQ ID NO.5, and comprises an arsenic-induced promoter system (1-492 bp), an ice nucleoprotein N-terminal domain sequence (599-1171 bp), a fluorescent protein gene (1178-1885 bp), an arsenic binding protein gene (1892-2245 bp) and an rrBT1T2 (2252-2723 bp).

EXAMPLE 4 construction of As1-ICAR2 (A12) expression vector for arsenic-binding protein

In this example, the As1-ICAR2 (A12) vector was constructed in order to obtain an expression vector in which the expression of the arsenic-specific binding protein ArsR2 was controlled by the Pars1 arsenic-inducible promoter, by replacing the ArsR1 gene fragment expressed by arsenic induction in the As1-ICAR (A11) vector with the ArsR2 gene fragment.

(1) Preparation of a linearized vector: the SalI and PstI double-enzyme digestion As1-ICAR vector is adopted, and a target band is recovered and purified by gel electrophoresis.

(2) Preparation of a DNA fragment of interest: as2-GP carrier is taken As a template, and the arsenic binding protein ArsR2 gene fragment is amplified by PCR. Pfu DNA polymerase (Suzhou Hongxn Biotech Co., ltd.) was used, and the sequence of the upstream primer was 5'-CGGCATGGACGAGCTGTACAAGGTCGACATGTCATTTCTGTTACCCATCCAATTG-3' (SalI) and the sequence of the downstream primer was 5'-CTCTCATCCGCCAAAACAGAAGCTCTGCAGTTAACTGCAAATGTTCTTACTGTCCCCGGAAC-3' (PstI).

(3) Construction of As1-ICAR2 vector: a recombinant reaction system was prepared on ice, the total volume was 20. Mu.L, 10. Mu.L (volume ratio 1:1) of the recombinant reaction system containing the target DNA fragment and the linearized vector and 10. Mu.L of 2 Xrecombinase reaction solution (Hongxin Biotechnology Ltd., suzhou) were reacted at 50 ℃ for 60 minutes, and then cooled on ice.

(4) Transformation and identification: 10 μ L of the recombinant reaction solution was used to transform E.coli TOP10 competent cells, which were plated on LB agar plate containing ampicillin (50 μ g/ml, the same applies below), and cultured overnight at 37 ℃. Single colonies were picked up in liquid LB containing ampicillin (final ampicillin concentration 50. Mu.g/mL, volume 5 mL) and cultured overnight at 37 ℃ at 200-250 r/min. The overnight culture solution was taken to extract plasmid to obtain As1-ICAR2 vector (FIG. 5) for sequencing verification of sequence accuracy. The nucleotide sequence of the arsenic-induced promoter expression system of the vector A12 (As 1-ICAR 2) is shown As SEQ ID No.1, and the nucleotide sequence of the arsenic-binding protein gene is shown As SEQ ID No. 4; the nucleotide sequence of the expression cassette is shown in SEQ ID NO.7, and comprises an arsenic-induced promoter system (1-492 bp), an ice nucleoprotein N-terminal structural domain sequence (599-1171 bp), a fluorescent protein gene (1178-1885 bp), an arsenic-binding protein gene (1892-2245 bp) and rrBT1T2 (2252-2723 bp).

Example 5 construction of specific arsenic-removing engineering bacteria and detection of expression of fusion protein

The a11 vector, the a21 vector and the a12 vector constructed in the above examples were transferred to competent cells of e.coli Top10, e.coli DH5 α and e.coli BL21, respectively, and the transformed competent cells were spread on LB agar medium containing ampicillin, and the plate was left at room temperature until the liquid was absorbed. The plate is inverted and cultured at 37 ℃, and bacterial colonies can appear after 12 to 16 hours. The monoclonal colonies were picked up separately and cultured overnight at 37 ℃ in liquid LB medium containing ampicillin (200 rpm), 2% in the next morningThe cells were inoculated in a new liquid LB medium containing ampicillin and cultured with shaking at 37 ℃ to OD ₆₀₀ When the concentration of the arsenic in the bacterial liquid is 0.5-0.8, dividing each bacterial liquid into 2 parts, adding an arsenic inducer solution with the final concentration of 150 mu mol/L of arsenic into one part of bacterial liquid (induced bacteria), and adding sterilized deionized ultrapure water into the other part of bacterial liquid (non-induced bacteria) to replace the arsenic inducer solution. The time for adding the arsenic inducer is 0h, and the OD of the bacterial liquid is respectively detected when the inducer is added and the bacterial liquid is cultured for 6h ₆₀₀ And fluorescence intensity values, and the results of growth and fluorescence intensity detection of each strain are shown in fig. 6, fig. 7, fig. 8, fig. 9, fig. 10, and fig. 11, respectively.

OD of gene engineering bacteria constructed by respectively transforming A11 vector, A21 vector and A12 vector through comparing three host bacteria of E.coli Top10, E.coli DH5 alpha and E.coli BL21 under arsenic induction condition ₆₀₀ The values (which indicate growth) and fluorescence intensity values found OD of E.coli Top10 and E.coli DH 5. Alpha. Strains of A11, A21 and A12 carriers when 150. Mu.M arsenic was used to induce each of the engineered bacteria, as compared with the non-induced bacteria ₆₀₀ The values all show a remarkable reduction trend, but the growth curves of the E.coli BL21 strains of the three vectors are not greatly different from those of non-induced bacteria, and the fluorescence intensities of the E.coli BL21 induced strains of the three vectors are higher than those of the E.coli Top10 and E.coli DH5 alpha induced strains of the three vectors. Therefore, the E.coli BL21 strain is more suitable to be used as a host strain than the E.coli DH5 alpha strain and the E.coli TOP10 strain, and the E.coli BL21 strain is selected to be used as the host strain in subsequent experiments to construct the gene engineering strain for specifically removing arsenic.

Compared with the corresponding non-induced strains, the genetically engineered bacteria constructed by respectively transforming A11, A21 and A12 vectors with the three host bacteria of E.coli Top10, E.coli DH5 alpha and E.coli BL21 show obviously improved fluorescence characteristics, which indicates that the constructed fusion protein can be normally expressed and can exert normal fluorescence characteristics in the three strains. In addition, from the fluorescence intensity values of the induced strains, it can also be seen that OD was found in BL21 strain carrying A21 vector ₆₀₀ Under the condition that the value is lower than that of the BL21 strain carrying the A12 vector, the fluorescence intensity value of the BL21 strain carrying the A21 vector is higher, which shows that the fluorescence property of the fusion protein expressed by the BL21 strain carrying the A21 vector is obviously higher than that of the fusion protein expressed by the A12 vectorThe fusion protein of (1).

Example 6 detection of arsenic removing ability of engineering bacteria

E.coli BL21 (A11-BL 21) containing A11 vector, E.coli BL21 (A21-BL 21) containing A21 vector and E.coli BL21 (A12-BL 21) containing A12 vector were selected as single clones, cultured overnight, inoculated at 2% ratio in LB medium containing ampicillin, and OD of the engineered bacteria was cultured ₆₀₀ When the concentration is about 0.6 to 1.0, the cells are retained by centrifugation, suspended in arsenic-containing solutions of different concentrations (10, 40, 160. Mu. Mol/L), cultured at 30 ℃ for 1 hour, centrifuged, and the arsenic content in the supernatant is measured. The arsenic content in the supernatant is lower than that in the solution before culture, which proves that the engineering bacteria have the function of adsorbing and removing arsenic (BL 21 empty bacteria without plasmids and BL21 bacteria with pUC57 plasmids are used as controls), the efficiency of removing arsenic is represented by the amount of adsorbed arsenic per gram of dry cells, and the arsenic content in the supernatant is determined by inductively coupled plasma mass spectrometry (ICP-MS).

The results are shown in FIG. 12, and show that compared with the control bacteria (BL 21 empty bacteria without plasmid and BL21 bacteria containing pUC57 plasmid), the arsenic removal effect of A11-BL21 and A21-BL21 in the three concentration groups is higher than that of the control bacteria (P < 0.05), the arsenic removal effect of A12-BL21 in the 10 and 40 μmol/L concentration groups is higher than that of the two control bacteria (P < 0.05), but the arsenic removal effect in the 160 μmol/L concentration group is not statistically different from that of the two control bacteria.

Compared with the arsenic removal effects of A11-BL21, A21-BL21 and A12-BL21, the arsenic removal effects of A21-BL21 in the concentration groups of 40 and 160 mu mol/L are higher than those of A11-BL21 bacteria (P is less than 0.05) except that the arsenic removal effects of A21-BL21 in the concentration group of 10 mu mol/L and A11-BL21 are not statistically different; the arsenic removal effect of A21-BL21 in the three concentration groups is higher than that of A12-BL21 bacteria (P < 0.05). The results show that the effect of adsorbing and removing arsenic of A21-BL21 is obviously better than that of A11-BL21 and A12-BL21.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> animal institute of Chinese academy of sciences of China institute of environmental sciences

<120> engineering bacterium for specifically removing arsenic, construction method and application thereof

<130> KHP201113793.7

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 492

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaattccaag ttatctcacc taccttaagg taatagtgtg attaatcata tgcgtttttg 60

gttatgtgtt gtttgactta atatcagagc cgagagatac ttgttttcta caaaggagag 120

ggaaatgttg caactaacac cacttcagtt atttaaaaac ctgtccgatg aaacccgttt 180

gggtatcgtg ttgttgctca gggagatggg agagttgtgc gtgtgtgatc tttgcatggc 240

actggatcaa tcacagccca aaatatcccg tcatctggcg atgctacggg aaagtggaat 300

ccttctggat cgtaaacagg gaaaatgggt tcactaccgc ttatcaccgc atattccttc 360

atgggctgcc cagattattg agcaggcctg gttaagccaa caggacgacg ttcaggtcat 420

cgcacgcaag ctggcttcag ttaactgctc cggtagcagt aaggctgtct gcatctaaaa 480

aatttgcctg aa 492

<210> 2

<211> 451

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttaccttcct ctgcacttac acattcgtta agtcatatat gtttttgact tatccgcttc 60

gaagagagac actacctgca acaatcagga gcgcaatatg tcatttctgt tacccatcca 120

attgttcaaa attcttgctg atgaaacccg tctgggcatc gttttactgc tcagcgaact 180

gggagagtta tgcgtctgcg atctctgcac tgctctcgac cagtcgcagc ccaagatctc 240

ccgccacctg gcattgctgc gtgaaagcgg gctattgctg gaccgcaagc aaggtaagtg 300

ggttcattac cgcttatcac cgcatattcc agcatgggcg gcgaaaatta ttgatgaggc 360

ctggcgatgt gaacaggaaa aggttcaggc gattgtccgc aacctggctc gacaaaactg 420

ttccggggac agtaagaaca tttgcagtta a 451

<210> 3

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgttgcaac taacaccact tcagttattt aaaaacctgt ccgatgaaac ccgtttgggt 60

atcgtgttgt tgctcaggga gatgggagag ttgtgcgtgt gtgatctttg catggcactg 120

gatcaatcac agcccaaaat atcccgtcat ctggcgatgc tacgggaaag tggaatcctt 180

ctggatcgta aacagggaaa atgggttcac taccgcttat caccgcatat tccttcatgg 240

gctgcccaga ttattgagca ggcctggtta agccaacagg acgacgttca ggtcatcgca 300

cgcaagctgg cttcagttaa ctgctccggt agcagtaagg ctgtctgcat ctaa 354

<210> 4

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgtcatttc tgttacccat ccaattgttc aaaattcttg ctgatgaaac ccgtctgggc 60

atcgttttac tgctcagcga actgggagag ttatgcgtct gcgatctctg cactgctctc 120

gaccagtcgc agcccaagat ctcccgccac ctggcattgc tgcgtgaaag cgggctattg 180

ctggaccgca agcaaggtaa gtgggttcat taccgcttat caccgcatat tccagcatgg 240

gcggcgaaaa ttattgatga ggcctggcga tgtgaacagg aaaaggttca ggcgattgtc 300

cgcaacctgg ctcgacaaaa ctgttccggg gacagtaaga acatttgcag ttaa 354

<210> 5

<211> 2723

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gaattccaag ttatctcacc taccttaagg taatagtgtg attaatcata tgcgtttttg 60

gttatgtgtt gtttgactta atatcagagc cgagagatac ttgttttcta caaaggagag 120

ggaaatgttg caactaacac cacttcagtt atttaaaaac ctgtccgatg aaacccgttt 180

gggtatcgtg ttgttgctca gggagatggg agagttgtgc gtgtgtgatc tttgcatggc 240

actggatcaa tcacagccca aaatatcccg tcatctggcg atgctacggg aaagtggaat 300

ccttctggat cgtaaacagg gaaaatgggt tcactaccgc ttatcaccgc atattccttc 360

atgggctgcc cagattattg agcaggcctg gttaagccaa caggacgacg ttcaggtcat 420

cgcacgcaag ctggcttcag ttaactgctc cggtagcagt aaggctgtct gcatctaaaa 480

aatttgcctg aaggatccga attccaagtt atctcaccta ccttaaggta atagtgtgat 540

taatcatatg cgtttttggt tatgtgttgt ggatccaaga aggagatata ccatggcaat 600

gactctcgac aaggcgttgg tgctgcgtac ctgtgcaaat aacatggccg atcactgcgg 660

ccttatatgg cccgcgtccg gcacggtgga atccagatac tggcagtcaa ccaggcggca 720

tgagaatggt ctggtcggtt tactgtgggg cgctggaacc agcgcttttc taagcgtgca 780

cgccgatgct cgatggattg tctgtgaagt tgccgttgca gacatcatca gtctggaaga 840

gccgggaatg gtcaagtttc cacgggccga ggtggttcat gtcggcgaca ggatcagcgc 900

gtcacacttc atttcggcac gtcaggccga ccctgcgtca acgtcaacgt caacgtcaac 960

gtcaacgtta acgccaatgc ctacggccat acccacgccc atgcctgcgg tagcaagtgt 1020

cacgttaccg gtggccgaac aggcccgtca cgaagtgttc gatgtcgcgt cggtcagcgc 1080

ggctgccgcc ccagtaaaca ccctgccggt gacgacgccg cagaatttgc agaccgccac 1140

ttacggcagc acgttgagtg gcgacaatca cctcatgatg gtgagcaagg gcgaggagga 1200

taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa 1260

cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac 1320

cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc 1380

tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt 1440

gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg 1500

cgtggtgacc gtgacccagg actcctccct ccaggacggc gagttcatct acaaggtgaa 1560

gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga cgatgggctg 1620

ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca 1680

gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc 1740

caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc 1800

ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac 1860

cggcggcatg gacgagctgt acaaggtcga catgttgcaa ctaacaccac ttcagttatt 1920

taaaaacctg tccgatgaaa cccgtttggg tatcgtgttg ttgctcaggg agatgggaga 1980

gttgtgcgtg tgtgatcttt gcatggcact ggatcaatca cagcccaaaa tatcccgtca 2040

tctggcgatg ctacgggaaa gtggaatcct tctggatcgt aaacagggaa aatgggttca 2100

ctaccgctta tcaccgcata ttccttcatg ggctgcccag attattgagc aggcctggtt 2160

aagccaacag gacgacgttc aggtcatcgc acgcaagctg gcttcagtta actgctccgg 2220

tagcagtaag gctgtctgca tctaactgca gagcttctgt tttggcggat gagagaagat 2280

tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 2340

tcccggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 2400

tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gccaactgcc aggcatcaaa 2460

taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 2520

acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 2580

ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 2640

ccatcctgac ggatggcctt tttgcgtttc tacaaactct ttgtttattt ttctaaatac 2700

attcaaatat gtatccgctc atg 2723

<210> 6

<211> 2684

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttaccttcct ctgcacttac acattcgtta agtcatatat gtttttgact tatccgcttc 60

gaagagagac actacctgca acaatcagga gcgcaatatg tcatttctgt tacccatcca 120

attgttcaaa attcttgctg atgaaacccg tctgggcatc gttttactgc tcagcgaact 180

gggagagtta tgcgtctgcg atctctgcac tgctctcgac cagtcgcagc ccaagatctc 240

ccgccacctg gcattgctgc gtgaaagcgg gctattgctg gaccgcaagc aaggtaagtg 300

ggttcattac cgcttatcac cgcatattcc agcatgggcg gcgaaaatta ttgatgaggc 360

ctggcgatgt gaacaggaaa aggttcaggc gattgtccgc aacctggctc gacaaaactg 420

ttccggggac agtaagaaca tttgcagtta aggatcctta ccttcctctg cacttacaca 480

ttcgttaagt catatatgtt tttgacttat ccgcttcgaa gagagacact aggatccaag 540

aaggagatat accatggcaa tgactctcga caaggcgttg gtgctgcgta cctgtgcaaa 600

taacatggcc gatcactgcg gccttatatg gcccgcgtcc ggcacggtgg aatccagata 660

ctggcagtca accaggcggc atgagaatgg tctggtcggt ttactgtggg gcgctggaac 720

cagcgctttt ctaagcgtgc acgccgatgc tcgatggatt gtctgtgaag ttgccgttgc 780

agacatcatc agtctggaag agccgggaat ggtcaagttt ccacgggccg aggtggttca 840

tgtcggcgac aggatcagcg cgtcacactt catttcggca cgtcaggccg accctgcgtc 900

aacgtcaacg tcaacgtcaa cgtcaacgtt aacgccaatg cctacggcca tacccacgcc 960

catgcctgcg gtagcaagtg tcacgttacc ggtggccgaa caggcccgtc acgaagtgtt 1020

cgatgtcgcg tcggtcagcg cggctgccgc cccagtaaac accctgccgg tgacgacgcc 1080

gcagaatttg cagaccgcca cttacggcag cacgttgagt ggcgacaatc acctcatgat 1140

ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt 1200

gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg 1260

cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt 1320

cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc 1380

cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt 1440

gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tccaggacgg 1500

cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat 1560

gcagaagaag acgatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc 1620

cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga 1680

ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa 1740

catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg 1800

cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaaggtcg acatgttgca 1860

actaacacca cttcagttat ttaaaaacct gtccgatgaa acccgtttgg gtatcgtgtt 1920

gttgctcagg gagatgggag agttgtgcgt gtgtgatctt tgcatggcac tggatcaatc 1980

acagcccaaa atatcccgtc atctggcgat gctacgggaa agtggaatcc ttctggatcg 2040

taaacaggga aaatgggttc actaccgctt atcaccgcat attccttcat gggctgccca 2100

gattattgag caggcctggt taagccaaca ggacgacgtt caggtcatcg cacgcaagct 2160

ggcttcagtt aactgctccg gtagcagtaa ggctgtctgc atctaactgc agagcttctg 2220

ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 2280

tctgataaaa cagaatttgc ctcccggcag tagcgcggtg gtcccacctg accccatgcc 2340

gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 2400

agccaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 2460

ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 2520

tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 2580

ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 2640

tttgtttatt tttctaaata cattcaaata tgtatccgct catg 2684

<210> 7

<211> 2723

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaattccaag ttatctcacc taccttaagg taatagtgtg attaatcata tgcgtttttg 60

gttatgtgtt gtttgactta atatcagagc cgagagatac ttgttttcta caaaggagag 120

ggaaatgttg caactaacac cacttcagtt atttaaaaac ctgtccgatg aaacccgttt 180

gggtatcgtg ttgttgctca gggagatggg agagttgtgc gtgtgtgatc tttgcatggc 240

actggatcaa tcacagccca aaatatcccg tcatctggcg atgctacggg aaagtggaat 300

ccttctggat cgtaaacagg gaaaatgggt tcactaccgc ttatcaccgc atattccttc 360

atgggctgcc cagattattg agcaggcctg gttaagccaa caggacgacg ttcaggtcat 420

cgcacgcaag ctggcttcag ttaactgctc cggtagcagt aaggctgtct gcatctaaaa 480

aatttgcctg aaggatccga attccaagtt atctcaccta ccttaaggta atagtgtgat 540

taatcatatg cgtttttggt tatgtgttgt ggatccaaga aggagatata ccatggcaat 600

gactctcgac aaggcgttgg tgctgcgtac ctgtgcaaat aacatggccg atcactgcgg 660

ccttatatgg cccgcgtccg gcacggtgga atccagatac tggcagtcaa ccaggcggca 720

tgagaatggt ctggtcggtt tactgtgggg cgctggaacc agcgcttttc taagcgtgca 780

cgccgatgct cgatggattg tctgtgaagt tgccgttgca gacatcatca gtctggaaga 840

gccgggaatg gtcaagtttc cacgggccga ggtggttcat gtcggcgaca ggatcagcgc 900

gtcacacttc atttcggcac gtcaggccga ccctgcgtca acgtcaacgt caacgtcaac 960

gtcaacgtta acgccaatgc ctacggccat acccacgccc atgcctgcgg tagcaagtgt 1020

cacgttaccg gtggccgaac aggcccgtca cgaagtgttc gatgtcgcgt cggtcagcgc 1080

ggctgccgcc ccagtaaaca ccctgccggt gacgacgccg cagaatttgc agaccgccac 1140

ttacggcagc acgttgagtg gcgacaatca cctcatgatg gtgagcaagg gcgaggagga 1200

taacatggcc atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa 1260

cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac 1320

cgccaagctg aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc 1380

tcagttcatg tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactactt 1440

gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg 1500

cgtggtgacc gtgacccagg actcctccct ccaggacggc gagttcatct acaaggtgaa 1560

gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga cgatgggctg 1620

ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca 1680

gaggctgaag ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc 1740

caagaagccc gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc 1800

ccacaacgag gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac 1860

cggcggcatg gacgagctgt acaaggtcga catgtcattt ctgttaccca tccaattgtt 1920

caaaattctt gctgatgaaa cccgtctggg catcgtttta ctgctcagcg aactgggaga 1980

gttatgcgtc tgcgatctct gcactgctct cgaccagtcg cagcccaaga tctcccgcca 2040

cctggcattg ctgcgtgaaa gcgggctatt gctggaccgc aagcaaggta agtgggttca 2100

ttaccgctta tcaccgcata ttccagcatg ggcggcgaaa attattgatg aggcctggcg 2160

atgtgaacag gaaaaggttc aggcgattgt ccgcaacctg gctcgacaaa actgttccgg 2220

ggacagtaag aacatttgca gttaactgca gagcttctgt tttggcggat gagagaagat 2280

tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc 2340

tcccggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg 2400

tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gccaactgcc aggcatcaaa 2460

taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga 2520

acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc 2580

ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg 2640

ccatcctgac ggatggcctt tttgcgtttc tacaaactct ttgtttattt ttctaaatac 2700

attcaaatat gtatccgctc atg 2723

<210> 8

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aactgcagag cttctgtttt ggcggatg 28

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgcatgcca tgagcggata catatttgaa tg 32

<210> 10

<211> 50

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgttgtggat ccaagaagga gatataccat ggcaatgcgt aaaggcgaag 50

<210> 11

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tctctcatcc gccaaaacag aagctctgca gtcatttgta cagttcatc 49

<210> 12

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aggaattccc gcggttacct tcctctgcac ttacac 36

<210> 13

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgacggatcc ttaactgcaa atgttcttac tgtcccc 37

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgacggatcc ttaactgcaa atgttcttac tgtcccc 37

<210> 15

<211> 62

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

ctctcatccg ccaaaacaga agctctgcag ttaactgcaa atgttcttac tgtccccgga 60

ac 62

Claims

1. The engineering bacteria for specifically removing arsenic is characterized in that the engineering bacteria is an escherichia coli BL21 strain carrying a vector containing an arsenic binding protein expression cassette, and the sequence of the expression cassette is shown as SEQ ID No. 6.

2. The engineered bacterium of claim 1, wherein the vector is a pUC series vector, a pBR322 series vector, or a pACYC series vector.

3. The method for constructing engineering bacteria according to claim 1 or 2, wherein the vector carrying the expression cassette is introduced into Escherichia coli BL21 strain.

4. A preparation comprising the engineered bacterium according to claim 1 or 2.

5. Use of the engineered bacterium of claim 1 or 2 or the formulation of claim 4 in the removal or recovery of arsenic.

6. Use of the engineered bacterium of claim 1 or 2 or the formulation of claim 4 in remediation of arsenic contamination of an environment.