BACTERIAL CELLULOSE-BASED BIOSENSOR AND USE THEREOF

Abstract

The invention provides a bacterial cellulose-based biosensor, including bacterial cellulose (BC) and a cell presenting a cellulose-binding module CBM2a on the surface. The cell is attached to BC through CBM2a. The cell expresses CBM2a by using pETDuet-tac as a vector. The pETDuet-tac is obtained by replacing two T7 promoters on the vector pETDuet by tac promoters, that is, an upstream first tac promoter and a downstream second tac promoter. The pETDuet-tac includes a gene encoding a fluorescent protein downstream of the first tac promoter and a gene encoding CBM2a presented on the surface downstream of the second tac promoter. By the BC-based biosensor, efficient and specific immobilization of cells on the BC matrix is enabled, the biological activity of cells is maintained, the fluorescence signal output is enhanced, and sufficient pores are provided for the entry and exit of a detected substance, thereby significantly improving the detection sensitivity.

Claims

1. A bacterial cellulose-based biosensor, comprising bacterial cellulose and a cell presenting a cellulose-binding module on the surface, wherein the cellulose-binding module specifically binds to crystalline region of cellulose, and the cell is attached to the bacterial cellulose through the cellulose-binding module, wherein the cellulose-binding module is CBM2a; the cell is a recombinant strain expressing the cellulose-binding module by using pETDuet-tac as a vector, wherein the pETDuet-tac is a vector obtained by replacing two T7 promoters on the vector pETDuet by two tac promoters, that is, an upstream first tac promoter and a downstream second tac promoter; and the pETDuet-tac comprises, a gene encoding a fluorescent protein downstream of the first tac promoter and a gene encoding the cellulose-binding module presented on the surface downstream of the second tac promoter; the first tac promoter is replaced by a promoter inducible by a substance to be tested, which affects the transcription of the downstream fluorescent protein coding gene in the presence of the target compound.

2. The biosensor according to claim 1, wherein the gene encoding CBM2a presented on the surface has a sequence as shown in SEQ ID NO. 1.

3. The biosensor according to claim 1, wherein the promoter inducible by a substance to be tested is a promoter inducible by L-arabinose, a promoter inducible by a nitro compound, or a promoter inducible by a heavy metal.

4. The biosensor according to claim 3, wherein the promoter inducible by L-arabinose has a nucleotide sequence as shown in SEQ ID NO. 3.

5. The biosensor according to claim 1, wherein the recombinant strain is a recombinant E. coli strain.

6. A method for constructing a biosensor according to claim 1, comprising steps of: (1) ligating a gene encoding the cellulose-binding module presented on the surface and a gene encoding a fluorescent protein to the vector pETDuet-tac, to obtain the vector pETDuet-tac-EGFP-CBM, wherein the gene encoding the fluorescent protein is located downstream of the first tac promoter, and the gene coding the cellulose-binding module presented on the surface is located downstream of the second tac promoter; and the cellulose-binding module is CBM2a; and (2) using the constructed vector pETDuet-tac-EGFP-CBM as a template, replacing the first tac promoter by a promoter inducible by a substance to be tested, transforming the vector into a host strain to obtain a recombinant strain, and co-incubating the recombinant strain with the bacterial cellulose to obtain the biosensor.

7. Use of the biosensor according to claim 1 in the detection of a substance.

8. The use according to claim 7, wherein the substance comprises a monosaccharide, an explosive molecule or a heavy metal.

9. The use according to claim 7, wherein the detection of a substance comprises steps of: mixing and incubating the biosensor with a sample to be tested, and then detecting the fluorescence intensity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To make the disclosure of the present invention more comprehensible, the present invention will be further described in detail by way of specific embodiments of the present invention with reference the accompanying drawings, in which

[0033] FIG. 1 is an SDS-PAGE electrophoretogram, in which 1 indicates a soluble protein derived from recombinant E. coli BL21(DE3) containing the plasmid pETDuet-tac-CBM2a, and 2 indicates a soluble protein derived from native E. coli BL21(DE3);

[0034] FIG. 2 is an immunofluorescence micrograph of recombinant E. coli with CBM2a presented on its surface;

[0035] FIG. 3 is an SEM image showing the surface morphology of a bacterial cellulose (BC) carrier loaded with E. coli cells presenting CBM2a on the surface;

[0036] FIG. 4 is a fluorescence image of a flaky and a spherical BC-based fluorescence biosensor for detecting L-arabinose (320 mg/L), respectively;

[0037] FIG. 5 shows the relationship between L-arabinose concentration and fluorescence intensity; and

[0038] FIG. 6 shows a fluorescence image of a flaky and a spherical BC-based fluorescence biosensor for detecting L-arabinose in soil.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention will be further described below with reference to the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention; however, the present invention is not limited thereto.

[0040] The methods given in examples below are all conventional methods, unless it is otherwise stated. The materials and reagents used are all commercially available, unless it is otherwise stated.

Example 1

[0041] (1) By using the PCR technology, the gene, as shown in SEQ ID NO. 1, encoding CBM2a presented on the surface of E. coli BL21(DE3) was inserted into the plasmid pETDuet-tac (using pETDuet as a template, in which two T7 promoter were replaced by two tac promoters, and which were preserved in the laboratory). The plasmid was enzymatically cleaved with the endonucleases NdeI and KpnI, purified by using a PCR purification kit, and recovered. Then, the sequence as shown in SEQ ID NO. 1 was ligated to the vector pETDuet-tac at 16? C. overnight by using T4 ligase. The ligated product was transformed into competent E. coli DH5a cells, and the vector pETDuet-tac-CBM2a was obtained after verification by colony PCR and sequencing. The pETDuet-tac-CBM2a was transformed into the host strain E. coli BL21(DE3), to obtain recombinant cells presenting CBM2a on the surface. [0042] (2) A single colony of Acetobacter xylinum was statically cultured for 2 days in 50 mL of HS medium (containing glucose 40 g/L, yeast extract 5 g/L, peptone 5 g/L, Na.sub.2HPO.sub.4 2.7 g/L, and acetic acid 1.5 g/L) at 30? C., to prepare a seed culture. 5 mL of the seed culture was transferred to 100 mL of HS medium and cultured statically at 30? C. for 15 days, to prepare a bacterial cellulose (BC) membrane. [0043] (3) A single colony of recombinant E. coli BL21(DE3) cells presenting CBM2a on the surface obtained in Step (1) was picked up and seeded into 5 mL of Luria-Bertani broth (LB) medium containing 100 ?g/mL Amp, and cultured at 37? C. with shaking at 200 rpm for 8-12 hrs. 1 mL of the cell culture was seeded into a 500 mL shake flask containing 100 mL culture medium, and cultured at 37? C. with shaking at 200 rpm. When OD.sub.600 reached 0.6-0.8, IPTG (with a final concentration of 0.25 mM) and BC matrix (obtained in Step 2) were added, and the cells were cultured for 12 hrs at 25? C. with shaking at 150 rpm, to obtain a BC carrier loaded with recombinant E. coli cells. Then, the BC carrier was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.0) to obtain a bacterial cellulose (BC)-based biosensor. [0044] (4) To determine the ability of E. coli presenting CBM2a to bind to BC, the cells released from the BC carrier was determined by measuring the fluorescence intensity and OD.sub.600 of the solution incubated with BC carrier. BC loaded with E. coli cells was cultured in 50 mM potassium phosphate buffer (pH 7.0) in a rotary hybridization system at 4? C. Then, samples were collected from the solution at various time points for fluorescence and OD.sub.600 measurement. Using a 96-well cell culture plate, the fluorescence intensity of escaped E. coli cells and the duration of OD.sub.600 were measured on cytation 5 imaging reader. The fluorescence of EGFP expressed in cells was characterized by using excitation light with ?ex=480 nm and emission light with ?em=520 nm.

[0045] The result shows that after an external shearing force is continuously applied to the BC carrier loaded with cells for 60 hrs, the cells presenting CBM on the surface can still be closely bound to the BC carrier.

Example 2

[0046] (1) By using the PCR technology, a gene encoding CBM presented on the surface of E. coli BL21(DE3) that specifically binds to crystalline region of cellulose could be inserted into the plasmid pETDuet-tac. The plasmid was enzymatically cleaved with the endonucleases NdeI and KpnI, purified by using a PCR purification kit, and recovered. The gene encoding CBM presented on the surface of E. coli BL21(DE3) that specifically binds to the crystalline region of cellulose was ligated to the vector pETDuet-tac at 16? C. overnight by T4 ligase. The ligated product was transformed into competent E. coli DH5a cells, and the vector pETDuet-tac-CBM2a was obtained after verification by colony PCR and sequencing. The pETDuet-tac-CBM2a was transformed into the host strain E. coli BL21(DE3), to obtain recombinant cells presenting CBM2a on the surface.

[0047] (2) A single colony of Acetobacter xylinum was statically cultured for 2 days in 50 mL of HS medium (containing glucose 40 g/L, yeast extract 5 g/L, peptone 5 g/L, Na.sub.2HPO.sub.4 2.7 g/L, and acetic acid 1.5 g/L) at 30? C., to prepare a seed culture. 10 mL of the seed culture was then transferred to 100 mL of HS medium and cultured statically at 30? C. for 15 days, to prepare a BC membrane.

[0048] (3) A single colony of recombinant E. coli BL21(DE3) cells presenting CBM2a that specifically binds to crystalline region of cellulose on the surface obtained in Step (1) were picked up and seeded into 5 mL of Luria-Bertani broth (LB) medium containing 100 ?g/mL Amp, and cultured at 37? C. with shaking at 200 rpm for 8-12 hrs. 1 mL of the cell culture was seeded into a 500 mL shake flask containing 100 mL culture medium, and cultured at 37? C. with shaking at 200 rpm. When OD.sub.600 reached 0.6-0.8, IPTG (with a final concentration of 0.25 mM) and BC matrix (obtained in Step 2) were added, and the cells were cultured for 12 hrs at 25? C. with shaking at 150 rpm, to obtain a BC carrier loaded with recombinant E. coli cells. Then, the BC carrier was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.0) to obtain a BC-based biosensor.

Example 3

[0049] (1) By using the PCR technology, the gene, as shown in SEQ ID NO. 1, encoding CBM2a presented on the surface of E. coli BL21(DE3) could be inserted into the plasmid pETDuet-tac (using pETDuet as a template, in which two T7 promoter were replaced by two tac promoters, and which were preserved in the laboratory). The plasmid was enzymatically cleaved with the endonucleases NdeI and KpnI, purified by using a PCR purification kit, and recovered. Then, the sequence as shown in SEQ ID NO. 1 was ligated to the vector pETDuet-tac at 16? C. overnight by using T4 ligase. The ligated product was transformed into competent E. coli DH5a cells, and the vector pETDuet-tac-CBM2a was obtained after verification by colony PCR and sequencing. Using pETDuet-tac-CBM2a as a template, the sites were enzymatically cleaved with NcoI and EcoRI, and the above enzymatic cleavage and ligation steps were repeated to insert the green fluorescent protein coding gene as shown in SEQ ID NO. 2 into pETDuet-tac-CBM2a. The ligated product was transformed into competent E. coli DH5a, and the vector pETDuet-tac-EGFP-CBM2a was obtained after verification by colony PCR and sequencing. The pETDuet-tac-EGFP-CBM2a was transformed into the host strain E. coli BL21(DE3), to obtain recombinant chassis fluorescent cell.

[0050] (2) A single colony of Acetobacter xylinum was statically cultured for 2 days in 50 mL of HS medium (containing glucose 40 g/L, yeast extract 5 g/L, peptone 5 g/L, Na.sub.2HPO.sub.4 2.7 g/L, and acetic acid 1.5 g/L) at 30? C., to prepare a seed culture.

[0051] 10 mL of the seed culture and 220 mL of HS medium were transferred into a 250 mL flask, and incubated for 5 days at 30? C. with shaking at 150 rpm, to prepare spherical BC. 10 mL of the seed culture and 100 ml of HS medium were poured into a 250 mL flask, and statically incubated for 15 days at 30? C., to prepare flaky BC. 10 mL of the seed culture and 100 ml of HS medium were mixed and then injected into a silica tube, and then statically incubated for 10 days at 30? C., to prepare tubular BC.

[0052] (3) A single colony of fluorescent E. coli BL21(DE3) chassis cells obtained in Step (1) was picked up and seeded into 5 mL of Luria-Bertani broth (LB) medium containing 100 ?g/mL Amp, and cultured at 37? C. with shaking at 200 rpm for 8-12 hrs. 1 mL of the cell culture was seeded into a 500 mL shake flask containing 100 mL culture medium, and cultured at 37? C. with shaking at 200 rpm. When OD.sub.600 reached 0.6-0.8, IPTG (with a final concentration of 0.25 mM) and BC matrix (obtained in Step 2) were added, and the cells were cultured for 12 hrs at 25? C. with shaking at 150 rpm, to obtain a flaky and a spherical BC carrier loaded with recombinant E. coli cells respectively, depending on the shape of the BC matrix. Then, the BC carrier was thoroughly washed with 50 mM potassium phosphate buffer (pH 7.0).

Example 4

[0053] Using the vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 as a vector, inverse PCR amplification was using primers shown below, to delete the tac promoter (first tac promoter) regulating green fluorescent protein:

TABLE-US-00005 primer1(SEQIDNO.6): 5-CAATCGATCTCGATCCTCTACG-3; primer2(SEQIDNO.7): 5-TTTCACACAGGAAACAGTATC-3.

[0054] In addition, using plasmid pCAS as a template, PCR amplification was performed with the following primers to obtain a nucleic acid fragment as shown in SEQ ID NO. 3 and comprising P.sub.araBAD and AraC:

TABLE-US-00006 primer3(SEQIDNO.8): 5-TAGAGGATCGAGATCGATTGTTATGACAACTTGACGGCTACA TC-3; primer4(SEQIDNO.9): 5-GATACTGTTTCCTGTGTGAAAATGGAGAAACAGTAGAGAGTTG CG-3.

[0055] Then, the fragment was ligated by using ClonExpress II One Step Cloning Kit (purchased from Vazyme), and the tac promoter regulating green fluorescent protein in the vector pETDuet-tac-EGFP-CBM2a was replaced by the nucleic acid fragment as shown in SEQ ID NO. 3 and comprising arabinose promoter (P.sub.araBAD) and AraC. The ligated product was transformed into competent E. coli DH5? cells, and the vector pETDuet-araBAD-EGFP-CBM2a was obtained after verification by colony PCR and sequencing. The recombinant plasmid pETDuet-araBAD-EGFP-CBM2a was transformed into the host strain E. coli BL21(DE3), and the cells were attached to BC according to the method described in Example 1 to obtain an L-arabinose-inducible fluorescence biosensor.

Example 5

[0056] (1) The L-arabinose-inducible fluorescence biosensor was used to detect L-arabinose in aqueous solution. The specific method was as follows. The L-arabinose-inducible fluorescence biosensor prepared above was immersed respectively in L-arabinose solutions with various concentrations, and incubated at room temperature for 5 hrs. Then the fluorescence intensity was measured and fluorescence imaging was carried out to detect L-arabinose in the solutions.

[0057] The L-arabinose solution was prepared by diluting an L-arabinose stock solution serially with water to give a final concentration of 20 mg/L, 160 mg/L and 320 mg/L respectively.

[0058] (2) The L-arabinose-inducible fluorescence biosensor was used to detect L-arabinose in soil. The specific method was as follows. A required soil sample was prepared by mixing L-arabinose with soil at a dosage of 2.4 g L-arabinose/Kg soil. Then, the fluorescence biosensor was buried in the sample, and incubated at 37? C. for 24 hrs. Then fluorescence imaging was performed to detect L-arabinose in the soil.

[0059] The results show that the lowest detectable concentration is 15 mg/L.

Example 6

[0060] (1) The vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 was used as a vector, in which the tac promoter regulating green fluorescent protein in the vector pETDuet-tac-EGFP-CBM2a was replaced by the yqjF promoter. The vector pETDuet-yqjF-EGFP-CBM2a was obtained. The recombinant plasmid pETDuet-yqjF-EGFP-CBM2a was transformed into the host strain E. coli BL21(DE3), and the cells were attached to BC according to the method described in Example 3 to obtain a 2,4-dinitrotoluene (2,4-DNT) inducible fluorescence biosensor.

[0061] (2) The 2,4-DNT inducible fluorescence biosensor was used to detect 2,4-DNT in aqueous solution. The specific method was as follows. The 2,4-DNT inducible fluorescence biosensor prepared above was immersed respectively in 2,4-DNT solutions with various concentrations, and incubated at room temperature for 12 hrs. Then the fluorescence intensity was measured and fluorescence imaging was carried out to detect 2,4-DNT in the solutions.

[0062] The 2,4-DNT solution was prepared by diluting an 2,4-DNT stock solution serially with water to give a final concentration of 5 mg/L, 10 mg/L and 20 mg/L respectively.

[0063] (3) The 2,4-DNT inducible fluorescence biosensor was used to detect 2,4-DNT in soil. The specific method was as follows. A required soil sample was prepared by mixing 2,4-DNT with soil at a dosage of 0.24 g 2,4-DNT/Kg soil. Then, the fluorescence biosensor was buried in the sample, and incubated at 37? C. for 24 hrs. Then fluorescence imaging was performed to detect 2,4-DNT in the soil.

[0064] The results show that the lowest detectable concentration is 4 mg/L.

Example 7

[0065] (1) The vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 was used as a template, in which the tac promoter relating green fluorescent protein in the vector pETDuet-tac-EGFP-CBM2a was replaced by the znt promoter (comprising zntA promoter and zntR nucleic acid sequence). The vector pETDuet-znt-EGFP-CBM2a was obtained. The recombinant plasmid pETDuet-CBM2a-znt-EGFP was transformed into the host strain E. coli BL21(DE3), and the cells were attached to BC according to the method described in Example 3 to obtain a heavy metal-inducible fluorescence biosensor.

[0066] (2) The heavy metal-inducible fluorescence biosensor was used to detect a heavy metal in aqueous solution. The specific method was as follows. The heavy metal-inducible fluorescence biosensor prepared above was immersed respectively in heavy metal solutions with various concentrations, and incubated at room temperature for 24 hrs. Then the fluorescence intensity was measured and fluorescence imaging was carried out to detect a heavy metal in the solutions.

[0067] The heavy metal solution comprises Zn.sup.2+, Cd.sup.2+, or Hg.sup.2+. The preparation method was as follows. A Zn.sup.2+ stock solution was serially diluted with water to a final concentration of 20 mg/L, 100 mg/L and 300 mg/L respectively. A Cd.sup.2+ stock solution was serially diluted with water to a final concentration of 0.5 mg/L, 2.0 mg/L and 4.0 mg/L respectively. A Hg.sup.2+ stock solution was serially diluted with water to a final concentration of 0.004 mg/L, 0.016 mg/L and 0.06 mg/L, respectively.

[0068] (3) The heavy metal-inducible fluorescence biosensor was used to detect a heavy metal in soil. The specific method was as follows. A required soil sample was prepared by mixing a heavy metal respectively with soil at a dosage of 0.3 g Zn.sup.2/Kg soil, 4 mg Cd.sup.2+/Kg soil and 0.06 mg Hg.sup.2+/Kg. Then, the fluorescence biosensor was buried in the sample, and incubated at 37? C. for 24 hrs. Then fluorescence imaging was performed to detect a heavy metal in the soil.

[0069] The results show that the lowest detectable concentrations are Zn.sup.2+ 6 mg/L, Cd.sup.2+ 0.05 mg/L, and Hg.sup.2+ 0.004 mg/L.

Comparative Example 1

[0070] (1) The PCR technology was used, the vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 was used as a template, and the second tac promoter was replaced by the T7 promoter. The vector pETDuet-tac-EGFP-T7-CBM2a was obtained. Then an L-arabinose-inducible fluorescence biosensor was prepared following the steps in Example 4.

[0071] (2) To compare the expression of CBM2a fusion protein presented on the surface in the presence of different promoters, SDS-PAGE was used to detect the intracellular soluble protein, and the ability of E. coli cells presenting CBM2a to bind to BC was monitored by determining the fluorescence intensity and OD.sub.600 of the solution incubated with the BC carrier according to Step (4) in Example 1. Then, L-arabinose in aqueous solution was detected by the L-arabinose-inducible fluorescence biosensor following the steps in Example 5.

[0072] The results show that overexpressed soluble proteins are present in both cells with different promoters, and the intracellular protein content in cells with T7 promoter was about 20% higher than that in cells with tac promoter. The experiment of cells' binding ability to BC shows that both cells can bind to the BC carrier closely, and the binding performances are basically the same. However, the higher overexpression of the protein requires higher consumption of the resources and energy in the cell. The detection sensitivity of the L-arabinose-inducible fluorescence biosensor containing T7 promoter decreases (by a factor of 2.1), and the lowest detectable concentration of L-arabinose is 32 mg/L.

Comparative Example 2

[0073] (1) The PCR technology was used, the vector pETDuet-tac-EGFP-CBM2a constructed in Example 1 was used as a template, and CBM2a was replaced by CBM44. The vector pETDuet-tac-EGFP-CBM44 was obtained.

[0074] Specific gene sequence of CBM44 (SEQ ID NO. 10):

TABLE-US-00007 5-AAATTCAATTTTGAAGATGGAACACTAGGGGGCTTTACCACCTC TGGCACCAATGCGACCGGTGTTGTGGTGAACACCACTGAAAAAGCGT TTAAGGGTGAACGTGGTCTGAAGTGGACCGTCACGTCCGAGGGCGAG GGCACCGCTGAGCTCAAGCTGGACGGCGGTACGATCGTGGTGCCGGG TACGACGATGACATTCCGCATTTGGATTCCGAGCGGCGCGCCAATCG CCGCAATTCAACCGTACATTATGCCGCACACCCCGGATTGGAGCGAA GTTCTGTGGAACAGCACCTGGAAAGGTTATACCATGGTCAAAACTGA CGATTGGAACGAGATCACCTTGACCCTGCCGGAAGATGTTGACCCGA CGTGGCCGCAGCAAATGGGTATTCAGGTTCAGACCATCGACGAAGGT GAGTTCACCATCTACGTGGATGCGATCGACTGGTGA-3.

[0075] The pETDuet-tac-EGFP-CBM44 was transformed into the host strain E. coli BL21(DE3), to obtain recombinant cells presenting CBM44 on the surface. A BC-based biosensor was obtained following the method in Example 1.

[0076] (2) To determine the ability of E. coli presenting CBM44 to bind to BC, the cells released from the BC carrier was determined by measuring the fluorescence intensity and OD.sub.600 of the solution incubated with BC carrier following the method in Example 1(4).

[0077] The result shows that after an external shearing force is continuously applied to the BC carrier loaded with cells for 60 hrs, the cells presenting CBM44 on the surface escape, and cannot be closely bound to the BC carrier. [0078] (3) By using the constructed vector pETDuet-tac-EGFP-CBM44 as a template, the tac promoter was replaced by a nucleic acid fragment comprising arabinose promoter (P.sub.araBAD) and AraC following the method in Example 4. Finally, an L-arabinose-inducible fluorescence biosensor bound with cells presenting CBM44 on the surface was obtained. Then, L-arabinose in aqueous solution and soil was detected respectively according to the method in Example 5.

[0079] The results show that due to cell escape, the detection sensitivity of the L-arabinose-inducible fluorescence biosensor bound with cells presenting CBM44 on the surface decreases greatly. For example, L-arabinose could not be detected normally at 20 mg/L, and the accuracy was less than 60% at 320 mg/L.

[0080] Apparently, the above-described embodiments are merely examples provided for clarity of description, and are not intended to limit the implementations of the present invention. Other variations or changes can be made by those skilled in the art based on the above description. The embodiments are not exhaustive herein. Obvious variations or changes derived therefrom also fall within the protection scope of the present invention.