An engineered bacterium and an engineered biofilm capable of sensing and adsorbing multiple heavy metal ions

Abstract

The present application discloses an engineered bacterium and an engineered biofilm that sense and adsorb a variety of heavy metal ions. The genomes of the engineered bacteria and the engineered biofilm include metal sensing elements and metal adsorption elements. The metal sensing elements include mercury sensing elements, lead sensing elements, and copper sensing elements. The metal adsorption elements include fusion protein genes. The fusion protein is a fusion protein of amyloid protein and metal binding protein. The present application develops a biofilm living material that can detect and adsorb a variety of heavy metal ion pollutants in real time by designing the gene circuits of metal sensing elements and metal adsorption elements. The material can self-replicate, respond to external environmental signals, and have material functional properties. It is also applied to environmental remediation to address the limitations of traditional detection methods and traditional chemical remediation methods and promote the sustainable development of the human health ecological environment.

Description

Technical Field

[0001] The present application relates to the field of molecular biology technology, and in particular to the construction and application of an engineered bacterium and an engineered biofilm. Background Art

[0002] Heavy metals accumulate along the food chain, causing human poisoning, which is a global public health safety issue. Due to the toxicity, non-degradability, universal use, and difficulty in recycling of heavy metal ions, large-scale heavy metal pollution remediation has caused great difficulties. Therefore, monitoring the concentration of heavy metals in the environment is of great significance. Traditional instrument-based methods, such as high-pressure liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry, are widely used to detect and quantify pollutants in environmental samples such as wastewater and soil. However, due to the complexity of the sample preparation stage, these methods are costly and not time-effective. Although synthetic biology has made initial progress in the modification of microorganisms for environmental pollution detection and remediation, most of them can only target a single heavy metal ion and cannot monitor multiple heavy metal ions at the same time.

[0003] In addition, although scientists have made many attempts and efforts to repair heavy metal ions, there are still many challenges and limitations. For example, physical and chemical repairs are costly, cannot be applied on a large scale, require a lot of manpower and material resources for treatment, and are prone to secondary pollution. Biological remediation mainly includes plant remediation and microbial remediation. Plant remediation has a long cycle and is not suitable for areas with excessively high pollution concentrations. Microbial remediation has the characteristics of short time, quick effect, self-replication, and response to environmental changes. In particular, biofilms have good stress resistance and are a potential remediation method.

[0004] The use of synthetic biology to modify microorganisms for environmental pollution detection and remediation has the advantages of high efficiency, specificity, controllability and environmental friendliness, which has begun to attract the attention of scientific researchers and has made a series of progress. At present, various cell biosensors have been developed for the detection and remediation of heavy metal pollution in the environment. Although many biosensors based on microbial modification have appeared, these biosensors can often only detect and / or adsorb single heavy metal ions. The actual sewage environment is more complex, and there are usually multiple heavy metal ions that exceed the standard, so materials with only a single adsorption property cannot purify sewage into clean water. In short, the above-mentioned adsorption and detection materials have the disadvantages of single function, high cost and easy secondary pollution. Summary of the invention

[0005] The present application provides an engineered bacterium and an engineered biofilm, which can improve the problems of current adsorption and detection materials having single functions, high costs, and easy secondary contamination.

[0006] According to the engineered bacteria in the first embodiment of the present application, its genome includes metal sensing elements and metal adsorption elements, the metal sensing elements include mercury sensing elements, lead sensing elements and copper sensing elements, the metal adsorption elements include fusion protein genes, and the fusion protein is a fusion protein of amyloid protein and metal binding protein.

[0007] Optionally, in other embodiments of the present application, the engineered bacteria is deposited in China Center for Type Culture Collection, and the collection number of the collection center is CCTCC NO: M 2023464.

[0008] Optionally, in other embodiments of the present application, the engineered bacteria is selected from Bacillus subtilis.

[0009] Optionally, in other embodiments of the present application, the mercury sensing element includes a mercury sensing regulator and a mercury sensing promoter, the nucleotide sequence of the mercury sensing regulator includes the sequence shown in SEQ ID No.1, and the nucleotide sequence of the mercury sensing promoter includes the sequence shown in SEQ ID No.2; the lead sensing element includes a lead sensing regulator and a lead sensing promoter, the nucleotide sequence of the lead sensing regulator includes the sequence shown in SEQ ID No.3, and the nucleotide sequence of the lead sensing promoter includes the sequence shown in SEQ ID No.4; and the copper sensing element includes a copper sensing promoter, and the nucleotide sequence of the copper sensing promoter includes the sequence shown in SEQ ID No.5.

[0010] Optionally, in other embodiments of the present application, the mercury sensing element further includes a first fluorescent gene, the first fluorescent gene includes a sequence as shown in SEQ ID No.6; the lead sensing element further includes a second fluorescent gene, the second fluorescent gene includes a sequence as shown in SEQ ID No.7; and the copper sensing element further includes a third fluorescent gene, the third fluorescent gene includes a sequence as shown in SEQ ID No.8.

[0011] Optionally, in other embodiments of the present application, the fusion protein gene includes a sequence as shown in SEQ ID No.9.

[0012] The present application also provides an engineered biofilm, wherein the engineered biofilm comprises the engineered bacteria and their secretions.

[0013] The present application also provides a composite material for sensing and adsorbing heavy metal pollutants, wherein the composite material comprises the engineered bacteria or engineered biofilm.

[0014] The present application also provides a use of the engineered bacteria or engineered biofilm as a sensor and adsorbent in the bioremediation of heavy metal pollution.

[0015] The engineered bacteria and engineered biofilm according to the embodiments of the present application have at least the following technical effects:

[0016] This application aims to develop a biofilm functional living material that specifically senses and adsorbs multiple heavy metals by integrating multiple metal sensing and adsorption elements into Bacillus subtilis, realize heavy metal sensing microbial sensing and remediation, and improve the tolerance and remediation ability of microorganisms to complex pollutants. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is a schematic diagram of the construction and application of engineered bacteria and engineered biofilms;

[0019] Figure 2 It is a technical roadmap for sensing and adsorption of engineered bacteria and engineered biofilms;

[0020] Figure 3 It is a heavy metal-inducible integrative plasmid map;

[0021] Figure 4 It is the TasA-Mli-his heavy metal adsorption plasmid map;

[0022] Figure 5 The lead-sensing promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Pb in water environment 2+ Content diagram of

[0023] Figure 6 The copper-sensing promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Cu in water environment 2+ Content diagram of

[0024] Figure 7 The mercury-sensitive promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Hg in water environment 2+ Content diagram of

[0025] Figure 8 is the fluorescence confocal microscopy image of the control group;

[0026] Fig. 9 15μM Pb 2+The induced strain 1935△△bslA△△::tasA-Mli-his-BGR expresses blue fluorescent protein;

[0027] Fig.10 20 μM Cu 2+ Figure 1. Induced strain 1935△△bslA△△::tasA-Mli-his-BGR expressing green fluorescent protein;

[0028] Fig.11 is 2.5 μM Hg 2+ Figure 1. Induced strain 1935△△bslA△△::tasA-Mli-his-BGR expressing red fluorescent protein;

[0029] Fig.12 This is an evaluation diagram of the sensitivity of strain 1935△△bslA△△::tasA-Mli-his-BGR to lead ions when simultaneously detecting multiple heavy metal ions in water environments;

[0030] Fig.13 This is an evaluation diagram of the sensitivity of strain 1935△△bslA△△::tasA-Mli-his-BGR to copper ions when simultaneously detecting multiple heavy metal ions in water environments;

[0031] Fig.14 This is an evaluation diagram of the sensitivity of strain 1935△△bslA△△::tasA-Mli-his-BGR to mercury ions when simultaneously detecting multiple heavy metal ions in water environments;

[0032] Fig.15 The lead-sensing promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Pb in water environment 2+ Specificity map;

[0033] Fig.16 The copper-sensitive promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Cu in water environment. 2+ Specificity map;

[0034] Fig.17 The mercury-sensitive promoter of strain 1935△△bslA△△::tasA-Mli-his-BGR detects Hg in water environment 2+ Specificity map;

[0035] Fig.18 When strain 1935△△bslA△△::tasA-Mli-his-BGR detects multiple heavy metal ions simultaneously, the promoter PpbrR detects Pb in water environment 2+ Specificity;

[0036] Fig.19 When strain 1935△△bslA△△::tasA-Mli-his-BGR detects multiple heavy metal ions simultaneously, the promoter PcopA detects Cu in water environment. 2+ Specificity;

[0037] Fig. 20 When strain 1935△△bslA△△::tasA-Mli-his-BGR detects multiple heavy metal ions simultaneously, the promoter Pmer detects Hg in water environment 2+ Specificity;

[0038] Fig.21 is a Western-blot analysis of TasA-R fusion protein expression;

[0039] Fig. 22 is a TEM image of strain 1935△eps△bslA△tasA△sinR cultured in MSgg medium for 2 days;

[0040] Fig.23 is the TEM image of strain 1935△eps△bslA cultured in MSgg medium for 2 days;

[0041] Fig.24 is a TEM image of strain 1935△△bslA△△::tasA-Mli-his-BGR cultured in MSgg medium for 2 days;

[0042] Fig.25 At different concentrations of Pb 2+ Below, the adsorption rate of heavy metal ions by the recombinant biofilm;

[0043] Fig.26 At different concentrations of Hg 2+ Below, the adsorption rate of heavy metal ions by the recombinant biofilm;

[0044] Fig. 27 At different concentrations of Cu 2+ Below, the adsorption rate of heavy metal ions by the recombinant biofilm;

[0045] Fig.28 There are many heavy metal ions in the water (T1: 5μM Cu 2+ +25μM Pb 2+ +25μM Hg 2+ ), the adsorption rate of each heavy metal ion by the recombinant biofilm;

[0046] Fig.29 There are many heavy metal ions in the water (T2: 10μM Cu2+ +25μM Pb 2+ +25μM Hg 2+ ) when the reconstructed biofilm was subjected to the adsorption rate of various heavy metal ions. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

[0048] The present application provides an engineering bacterium that senses and adsorbs multiple heavy metals, the construction of an engineered biofilm and its application. The following are detailed descriptions. It should be noted that the description order of the following embodiments is not intended to limit the preferred order of the embodiments.

[0049] The experimental methods in the following examples where specific conditions are not specified are usually performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0050] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to this application. The preferred implementation methods and materials described herein are for demonstration purposes only and are not intended to limit the content of this application.

[0051] Unless otherwise specified, the reagents and materials involved in the text are commercially available, or can be prepared by those skilled in the art according to common knowledge.

[0052] Definition and explanation:

[0053] As used in this application, "nucleotide sequence" refers to the sequence of nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which can be single-stranded or double-stranded.

[0054] As used herein, "promoter" refers to a nucleic acid region located upstream of the translation start codon and involved in the recognition and binding of RNA polymerase and other transcription proteins.

[0055] As used in this application, "regulator" refers to a base sequence having a functional promoter and any associated transcription elements (eg, enhancer, CCAAT box, TATA box, SPI portion, etc.).

[0056] As used herein, "plasmid" refers to an autonomous circular DNA molecule capable of replicating in a cell.

[0057] As used herein, "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0058] An embodiment of the present application provides an engineered bacterium, wherein the genome of the engineered bacterium includes a metal sensing element and a metal adsorption element, wherein the metal sensing element includes a mercury sensing element, a lead sensing element, and a copper sensing element, and the metal adsorption element includes a fusion protein gene, and the fusion protein is a fusion protein of an amyloid protein and a metal binding protein.

[0059] At present, heavy metal instrument detection has problems such as long time consumption and high instrument cost, and microbial detection has problems such as single function, poor stress resistance, and narrow detection range. In response to the above problems, the engineered bacteria constructed in this application are designed to intelligently sense a variety of heavy metals, realize real-time detection of a variety of heavy metal ions, and have good specificity, sensitivity and a wide detection range. The current heavy metal adsorption methods have problems such as long time consumption, high cost, difficult recovery, and secondary pollution caused by chemical reagents. In response to the above problems, this application will use a safe and non-toxic microbial method for repair, and construct a recombinant biofilm with environmental tolerance and functional stability for the adsorption of a variety of heavy metals.

[0060] In order to solve the above problems, the present invention screens the heavy metal ion Pb 2+ , Cu 2+ , Hg 2+ The invention relates to an engineering bacterium containing heavy metal sensing and repair gene circuits (gene circuits are composed of gene elements, which regulate the expression of target genes according to given conditions. The gene circuits are connected to plasmids and transformed into bacteria so that the target genes can be expressed), and the engineered bacteria and engineered biofilms are used to sense heavy metal pollutants, emit fluorescence, form biofilms, and then adsorb pollutants. The biofilm living material formed by the engineered bacteria constructed in the present application has the characteristics of self-replication, sensing heavy metal ions, and in-situ adsorption, and has stronger environmental tolerance and robustness than free cells.

[0061] The engineered bacteria are deposited in China Center for Type Culture Collection, and the collection number of the collection center is CCTCC NO: M2023464.

[0062] In some embodiments of the present application, the engineered bacteria and engineered biofilm are selected from Bacillus subtilis. Since Bacillus subtilis is a non-pathogenic Gram-positive bacterium widely distributed in the environment, it is easy to survive in soil and corrupted organic matter, does not contain exotoxins and endotoxins, and is defined as a "generally recognized as safe strain (GRAS)" in industrial production and pharmaceuticals, and is easy to form a biofilm at the gas-liquid interface in the natural environment. Biofilm is a complex group structure containing substances such as living bacteria, extracellular polysaccharides, eDNA, lipids and proteins (TasA / BslA), and has the characteristics of resisting harsh environments such as acid resistance, alkali resistance, and salt resistance. In recent years, the robustness of biofilms has been used to transform biofilms in combination with engineering methods, so that they can be applied to the fields of environmental remediation and live catalysis. This application uses Bacillus subtilis as a chassis cell, designs a gene circuit for sensing and adsorbing heavy metals, develops a biofilm living material that can detect and adsorb a variety of heavy metal ion pollutants in real time, and applies it to environmental remediation.

[0063] In some embodiments of the present application, the mercury sensing element includes a mercury sensing regulator and a mercury sensing promoter, the nucleotide sequence of the mercury sensing regulator merR includes the sequence shown in SEQ ID No.1, and the nucleotide sequence of the mercury sensing promoter Pmer includes the sequence shown in SEQ ID No.2.

[0064] In some embodiments of the present application, the lead-sensitive element includes a lead-sensitive regulator and a lead-sensitive promoter, the nucleotide sequence of the lead-sensitive regulator pbrR includes the sequence shown in SEQ ID No.3, and the nucleotide sequence of the lead-sensitive promoter PpbrR includes the sequence shown in SEQ ID No.4.

[0065] In some embodiments of the present application, the copper-sensitive element includes a copper-sensitive promoter, and the nucleotide sequence of the copper-sensitive promoter PcopA includes the sequence shown in SEQ ID No.5.

[0066] In some embodiments of the present application, the mercury sensing element further includes a first fluorescent gene mCherry, and the first fluorescent gene includes a sequence as shown in SEQ ID No.6.

[0067] In some embodiments of the present application, the lead sensing element further includes a second fluorescent gene mtagBFP, and the second fluorescent gene includes a sequence as shown in SEQ ID No.7.

[0068] In some embodiments of the present application, the copper sensing element further includes a third fluorescent gene eGFP, and the third fluorescent gene includes a sequence as shown in SEQ ID No.8.

[0069] The heavy metal ions Pb 2+ , Cu 2+ and Hg 2+ The promoters PpbrR, PcopA, and Pmer were combined with the fluorescent protein genes mtagBFP, eGFP, and mCherry genes that do not interfere with each other to construct a gene circuit for sensing three heavy metal ions (lead, mercury, and copper ions). After verification by sequencing, the circuit was transformed into an engineered Bacillus subtilis to monitor the content of heavy metal ions in the environment in real time. Considering the stability of the detection element, the detection element was integrated into the genomic amyE site.

[0070] In some embodiments of the present application, the fusion protein gene tasA-mli-his includes a sequence as shown in SEQ ID No.9. Based on the Bacillus subtilis biofilm TasA display platform, the present application fuses the metal binding protein (Mli) with TasA for expression, displays the protein that adsorbs heavy metal ions on the surface of the bacteria, and makes it easier for the metal binding protein to approach and adsorb heavy metal ions. In order to achieve the expression of functional proteins without adding inducers and to realize the principle of adsorption, the present application will use the constitutive promoter of Bacillus subtilis itself to start the expression of biofilm fiber protein and the metal binding protein (TasA-R), and construct a Bacillus subtilis biofilm that continuously adsorbs heavy metal ions (lead, mercury, copper and other ions), thereby reducing the harm of heavy metal ions to the ecological environment. In order to ensure the expression stability of the fusion protein, the present application replaces the gene sequence in situ to the Bacillus subtilis tasAsinR site.

[0071] like Figure 1 and Figure 2 As shown, the heavy metal ions Pb 2+ , Cu 2+ and Hg 2+ The promoters PpbrR, PcopA and Pmer were combined with the fluorescent protein genes mtagBFP, eGFP and mCherry genes that do not interfere with each other to construct a gene circuit that simultaneously senses three heavy metal ions (lead, mercury, copper and other ions). When heavy metal ions exist in the environment, the strain expresses fluorescent protein; the metal binding protein is fused with TasA for expression, and the protein that adsorbs heavy metal ions is displayed on the surface of the bacteria, thereby adsorbing heavy metal ions in the environment.

[0072] Correspondingly, an embodiment of the present application also provides a composite material for sensing and adsorbing heavy metal pollutants, wherein the composite material includes the engineered bacteria and the engineered biofilm.

[0073] In addition, the embodiments of the present application also provide a use of the engineered bacteria and engineered biofilm as sensors and adsorbents in the bioremediation of heavy metal pollution.

[0074] The description of plasmids and strains in the examples of this application is detailed in Table 1 below:

[0075] Table 1

[0076]

[0077]

[0078]

[0079] The technical solution and beneficial effects of the present application are further explained below in conjunction with embodiments.

[0080] Embodiment 1: Engineered bacteria intelligently sense heavy metal ions such as lead, copper and mercury in water.

[0081] Construction of heavy metal inducible plasmids and heavy metal adsorption plasmids. Figure 3 The inducible integration plasmid is shown in Figure 2. The promoters PpbrR, PcopA and Pmer of the plasmid respond to heavy metal ions Pb 2+ , Cu 2+ and Hg 2+ After that, the fluorescent proteins mtagBFP, eGFP and mCherry were expressed. Figure 4 The figure shows the TasA-Mli-his heavy metal adsorption plasmid, which replaces the tasA-sinR site in situ in Bacillus subtilis to express the protein TasA-Mli-his that can adsorb heavy metal ions.

[0082] Constructed a system that can simultaneously respond to Pb 2+ , Cu 2+ , Hg 2+ The gene circuits pDG-pbrR-PpbrR-mtagBFP-PcopA-eGFP-merR-Pmer-mCherry for various heavy metal ions were integrated into the amyE site of strain 1935△eps△bslA to obtain 1935△eps△bslA△amyE::BGR, which can sense Pb 2+ , Cu 2+ and Hg 2+Then, blue, green or red fluorescence is emitted. Based on this strain, the TasA-Mli-his heavy metal adsorption plasmid is integrated into the tasA site to obtain the final strain 1935△eps△bslA△tasA△sinR::PtasA-tasA-mli-his△amyE::BGR (the name of this strain is 1935△△bslA△△::tasA-Mli-his-BGR unless otherwise specified in this patent).

[0083] To determine the detection range of heavy metal ions by the constructed engineered bacteria 1935△△bslA△△::tasA-Mli-his-BGR, a gradient concentration of 0-100 μM Pb 2+ and Cu 2+ The strain was induced overnight, centrifuged and resuspended in PBS buffer and standardized to OD600 nm ~ 1.0. The test found that strain 1935△△bslA△△::tasA-Mli-his-BGR detected Pb 2+ and Cu 2+ The range of fluorescence intensity was between 0.1 and 100 μM. When the concentration reached 100 μM, the fluorescence intensity was low. The maximum fluorescence value was reached at 15 and 20 μM respectively. Figure 5 and Figure 6 ). Due to Hg 2+ It is too toxic to the strain, so first use 0.01-4.5μM Hg 2+ Bacillus subtilis 1935△△bslA△△::tasA-Mli-his-BGR was induced overnight and the same detection method was used. The results showed that 1935△△bslA△△::tasA-Mli-his-BGR was resistant to Hg 2+ The detection range is 0.01~4μM, even when Hg 2+ When the induction concentration was as low as 0.01 μM, the microplate reader could still detect the cell response to Hg 2+ The red fluorescence ( Figure 7 ), and when Hg 2+ When the induction concentration was as high as 4.5 μM, the bacteria died, which may be due to its strong toxicity.

[0084] At the same time, the strain was observed using a confocal microscope to observe the expression of fluorescent protein to confirm its detection activity. Figures 8 to 11 As shown, compared with the strain without any heavy metals, 15 μM Pb 2+ , 20μM Cu 2+ and 2.5 μM Hg 2+ The heavy metal ion-induced engineered strains expressed the strongest blue, green and red fluorescence respectively, and the experimental results were consistent with the results of the ELISA instrument detection, indicating that the strain has a wide detection range.

[0085] Pb exists alone in the environment 2+ , Cu 2+ or Hg 2+ When the strain 1935△△bslA△△::tasA-Mli-his-BGR has good metal selectivity. However, in the actual water environment, the heavy metal pollution is complex and diverse. 2+ , Cu 2+ and Hg 2+ In order to study this issue, a final concentration of G2 (1 μM Pb 2+ +1μM Cu 2+ +0.1μM Hg 2+ )、G3(10μM Pb 2+ +10 μM Cu 2+ +1μM Hg 2+ )、G4(15μM Pb 2+ +20μM Cu 2+ +2.5μM Hg 2+ )、G5(20μM Pb 2+ +25 μM Cu 2+ +3μM Hg 2+ ), G1 was used as a blank control without any heavy metal ions, and the 1935△△bslA△△::tasA-Mli-his-BGR strain was induced to express overnight. Then the cells were collected and resuspended in PBS buffer, and the fluorescence intensity of each sample was measured. The results showed that the heavy metal-inducible promoters PpbrR, PcopA and Pmer were not affected by the induction of other heavy metal ions even in the same gene line, and the strain still had good detection sensitivity and could simultaneously detect three heavy metal ions in the environment ( Figure 12 to Figure 14 ).

[0086] After determining the concentration range of heavy metal ions detected by each promoter, in order to detect whether each promoter would be interfered by other heavy metal ions, the metal selectivity of the modified engineered strains was evaluated. 2+ , Cu 2+ , Hg 2+ 、Ni 2+ , Fe 2+ 、Cd 2+ 、Zn 2+ 、Ag 2+ 、Co 2+ Cr 2+Added to LB, induced 1935△△bslA△△::tasA-Mli-his-BGR strain to express overnight. Then the cells were collected and resuspended in PBS buffer, and the fluorescence intensity of each sample was measured. The results showed that only Pb 2+ In the presence of , the promoter PpbrR can efficiently express the blue fluorescent protein mtagBFP ( Fig.15 ). Meanwhile, in another set of experiments, 20 μM Cu 2+ , Pb 2+ , Hg 2+ 、Ni 2+ , Fe 2+ 、Cd 2+ 、Zn 2+ 、Ag 2+ 、Co 2+ or 2.5 μM Hg 2+ , Pb 2+ , Cu 2+ 、Ni 2+ , Fe 2+ 、Cd 2+ 、Zn 2+ 、Ag 2+ 、Co 2+ Inducing 1935△△△△::BGR strain, it was found that PcopA and Pmer were only found in Cu 2+ , Hg 2+ ions, the fluorescent gene can be effectively expressed and detected, indicating that the constructed strain has good specificity ( Figure 16-17 ).

[0087] To determine Pb 2+ , Cu 2+ , Hg 2+ When the three promoters coexist, will they interfere with the metal selection of the three promoters? The same detection method was used for strain 1935△△bslA△△::tasA-Mli-his-BGR ( Figure 18 to Figure 20 ). Select 6 irrelevant heavy metal ions Ni 2+ , Fe 2+ 、Zn 2+ 、Ag 2+ 、Co 2+ Cr 2+ As a positive control, 15 μM Pb was added to the six heavy metal ions. 2+ , 20μM Cu 2+ , 2.5μM Hg 2+ As the experimental group, the overnight induced strain 1935△△bslA△△::tasA-Mli-his-BGR was used. The test found that only when Pb 2+, Cu 2+ , Hg 2+ In order to effectively induce the strain 1935△△bslA△△::tasA-Mli-his-BGR to express fluorescent protein. 2+ , Cu 2+ , Hg 2+ When compared with other heavy metal ions, PpbrR, PcopA, and Pmer are still not affected by the interference induced by other heavy metals, and all have good metal selection specificity.

[0088] In summary, the Bacillus subtilis genetically engineered bacteria constructed in this application has good detection sensitivity and selective specificity for heavy metals, and has a wide detection range. The experimental results show that the heavy metal sensing engineered bacteria 1935△△bslA△△::tasA-Mli-his-BGR constructed in this application can detect 0.1-100μM Pb 2+ , 0.1-100 μM Cu 2+ and 0.01-4.0 μM Hg 2+ This detection range can be used to detect metal Pb in sewage 2+ , Cu 2+ , Hg 2+ Whether it complies with the emission standards (Pb first category pollutant emission limit 0.48μM, Hg first category pollutant emission limit 0.02μM, Cu first category pollutant emission limit 3.15μM).

[0089] Example 2: Evaluation of the ability of engineered biofilms to adsorb heavy metal ions.

[0090] In addition to integrating heavy metal sensing gene circuits, the engineered bacteria also integrates a biofilm expression system for heavy metal adsorption. The TasA fibrin protein in the Bacillus subtilis biofilm is resistant to harsh environments such as acids, alkalis, and detergents, and the TasA display platform is capable of displaying proteins of up to 600 amino acids. Therefore, this application attempts to fuse the metal sulfide binding protein (Mli) with TasA and display it on the surface of bacteria for the removal of heavy metals in water environments. To verify the successful display of the proteins TasA and TasA-Mli-his on the surface of Bacillus subtilis, each recombinant biofilm was cultured for 48 hours, and then Western-blot was used to analyze the expression of the fusion protein. Fig.21 As shown, by using the corresponding TasA antibody as the primary antibody and the universal antibody as the secondary antibody, it was found through color development reaction that TasA (28.73 kDa) and TasA-Mli-his (35.29 kDa) were close to their theoretical values ​​and were successfully expressed, which indicates that they are easily exposed to metal ions in the extracellular environment.

[0091] In order to verify that the TasA protein surface display system can effectively display heavy metal binding proteins, the constructed strain was cultured in MSgg medium for 3 days and then observed using transmission electron microscopy (TEM) and immunogold labeling. Figure 22 to Figure 24 As shown, compared with the control strain 1935△eps△bslA△tasA△sinR, TasA-Mli-his had obvious fiber formation, which preliminarily indicated that the fusion protein was successfully expressed and could be assembled into fibers displayed on the bacterial surface, which could be used to adsorb heavy metal ions in the environment.

[0092] In order to evaluate the effectiveness of the recombinant biofilm TasA-Mli-his in adsorbing heavy metals, three heavy metal ions Cu 2+ , Pb 2+ , Hg 2+ After the cultured recombinant biofilm was incubated with different heavy metal ion aqueous solutions, the cells were collected by centrifugation and adsorbed using HNO 3 After digestion, the concentration of heavy metal ions in the bacteria was determined by ICP-MS. Figure 25 to Figure 27 The results show the changes in the concentration of different heavy metal ion solutions after adsorption by the recombinant biofilm. The experimental results show that the recombinant biofilm has good adsorption performance for the three common environmental heavy metal pollutants tested. 2+ , Hg 2+ The ion concentration is 25-100 μM, Cu 2+ When the ion concentration was 5-100μM, the adsorption efficiency of the membrane decreased with the increase of metal ion concentration. The adsorption capacity of the recombinant biofilm TasA-Mli-His was higher than that of the control group TasA.

[0093] When 20 mg (dry weight) of engineered (recombinant) biofilm TasA-Mli-his was used to adsorb 25 μM Pb 2+ When the adsorption rate can reach 99.52%, the remaining Pb in the water 2+ The content was reduced to 0.12 μM, which was 2.01 times higher than that of the strain 1935△eps△bslA△tasA△sinR that did not produce a membrane. Fig.25 ). Similarly, 20 mg (dry weight) of the recombinant membrane adsorbed 25 μM Hg 2+ After adsorption, the adsorption rate is 99.98%, and the remaining Hg in the water 2+ The content was reduced to 0.005 μM, which was 1.17 times higher than that of the strain 1935△eps△bslA△tasA△sinR that did not produce a membrane ( Fig.26 ). 20 mg (dry weight) of recombinant biofilm adsorbed 5 μM Cu 2+After that, the adsorption rate was 99.56%, and the remaining Cu 2+ The concentration was reduced to 0.022 μM, which was 1.55 times higher than that of the strain 1935△eps△bslA△tasA△sinR that did not produce a membrane. Fig. 27 ).

[0094] The above experimental results show that the engineered biofilm we constructed has good removal performance for these three heavy metal ions. 2+ , Hg 2+ 25 μM, Cu 2+ When the concentration of metal ions in the water was 5 μM, after adsorption using a recombinant biofilm TasA-Mli-his with a dry weight of up to 20 mg, the concentrations of metal ions in the water were reduced to the dischargeable standard (DB31 / 199-2018, Pb 0.48 μM, Hg 0.02 μM, Cu 3.15 μM).

[0095] Considering that polluted water may contain multiple heavy metal pollutants, this application further studied the efficiency of reconstructed biofilm in removing multiple heavy metal ions simultaneously. Here we used three common heavy metal ion pollutants for evaluation and selected two groups of different heavy metal concentrations, 5μM Cu 2+ +25μM Pb 2+ +25μM Hg 2+ (T1), 10 μM Cu 2+ +25μMPb 2+ +25μM Hg 2+ (T2), such as Figure 28-29 As shown in the figure, all metal ions were effectively captured by the recombinant membrane TasA-Mli-his. 20 mg of bacterial TasA-Mli-his 2+ , Hg 2+ The adsorption effect is excellent. When the concentration is 25μM, the adsorption rate is maintained at 99.30%-99.51% and 99.46%-99.54% respectively, which almost achieves 100% removal of polluting heavy metal ions in water. After adsorption, the metal ion Pb in the water is 2+ , Hg 2+ The concentrations are all lower than the sewage discharge standards.

[0096] This application designs genetic circuits of metal sensing elements and metal adsorption elements to develop biofilm living materials that can detect and adsorb a variety of heavy metal ion pollutants in real time. The material can self-replicate, respond to external environmental signals, and have material functional properties. It is applied to environmental remediation to overcome the limitations of traditional detection methods and traditional chemical remediation methods and promote the sustainable development of human health and ecological environment.

[0097] The above is a detailed introduction to an engineered bacteria and an engineered biofilm and their applications provided in the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technical personnel in this field, according to the ideas of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. An engineered bacterium, characterized in that: The genome of the engineered bacteria includes metal sensing elements and metal adsorption elements, wherein the metal sensing elements include mercury sensing elements, lead sensing elements and copper sensing elements, and the metal adsorption elements include fusion protein genes, wherein the fusion protein is a fusion protein of amyloid protein and metal binding protein. Wherein the engineered bacteria is selected from Bacillus subtilis; The mercury sensing element includes a mercury sensing regulator, a mercury sensing promoter and a first fluorescent gene, the nucleotide sequence of the mercury sensing regulator is the sequence shown in SEQ ID No.1, the nucleotide sequence of the mercury sensing promoter is the sequence shown in SEQ ID No.2, and the first fluorescent gene is the sequence shown in SEQ ID No.6; The lead sensing element comprises a lead sensing regulator, a lead sensing promoter and a second fluorescent gene, wherein the nucleotide sequence of the lead sensing regulator is the sequence shown in SEQ ID No.3, the nucleotide sequence of the lead sensing promoter is the sequence shown in SEQ ID No.4, and the second fluorescent gene is the sequence shown in SEQ ID No.7; The copper-sensing element comprises a copper-sensing promoter and a third fluorescent gene. The nucleotide sequence of the copper-sensing promoter is the sequence shown in SEQ ID No.5, and the third fluorescent gene is the sequence shown in SEQ ID No.

8.

2. The engineered bacteria according to claim 1, characterized in that The engineered bacterial strain is deposited in China Center for Type Culture Collection, and the collection number of the collection center is CCTCC NO: M 2023464.

3. An engineered biofilm, characterized in that: The engineered biofilm comprises the engineered bacteria as claimed in claim 1 and its secretions.

4. A composite material for sensing and adsorbing heavy metal pollutants, characterized in that: The composite material comprises the engineered bacteria or engineered biofilm as described in claim 1 or 2.

5. Use of the engineered bacteria or engineered biofilm as claimed in claim 1 or 2 as a sensor and adsorbent in the bioremediation of heavy metal pollution.

Images