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A bacterial cellulose-based biosensor and its application

A biosensor, bacterial cellulose technology, applied in the fields of genetic engineering, nanomaterials and biosensors, and molecular biology, can solve the problems of interfering analyte monitoring, bacteria cannot continue to adhere for a long time, and the preparation process is time-consuming, and achieves high efficiency. and specific fixation, rapid whole-cell loading

Active Publication Date: 2022-08-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

BC is widely used in gas sensors, surface acoustic wave humidity sensors, electrochemical sensors, etc. However, when used in biosensors, due to the large pores between BC fibers, bacteria cannot continue to adhere for a long time, and escape will occur during the sensing process. , limiting the application of BC as a biosensor
[0004] Kim et al. (2019) developed a P(HEMA-co-HAETC) hydrogel-based bacterial sensor, which first prepared P(HEMA-co-HAETC) hydrogel beads using electrospray, and then passed a 12-hour Incubation to load cells onto hydrogel beads, the preparation process is time-consuming and requires the use of specialized equipment; Drachuk et al. method in BC materials, but this method introduces BC-producing bacteria into the biosensing system and interferes with the monitoring of analytes; in addition, Yoetz Kopelman et al. (2016) proposed a method to form positive charge, to enhance the affinity of negatively charged bacteria to the support immobilization method, although this method proved to be feasible as a whole-cell biosensor, it will cause non-specific binding of cells to the matrix

Method used

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  • A bacterial cellulose-based biosensor and its application
  • A bacterial cellulose-based biosensor and its application
  • A bacterial cellulose-based biosensor and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] (1) Using PCR technology, insert the gene SEQID NO.1 that can display CBM2a on the surface of E. coli E.coli BL21 (DE3) into the plasmid pETDuet-tac (with pETDuet as the template, and replace the two T7 promoters on it with tac promoter, laboratory deposit). It was digested with endonucleases NdeI and KpnI, and purified and recovered by PCR purification kit. Then, T4 ligase was used to ligate overnight at 16°C, and SEQ ID NO. 1 was ligated with the vector pETDuet-tac. The ligation product was transformed into competent cells E.coli DH5α, and the colony PCR and sequencing were verified to obtain the vector pETDuet-tac-CBM2a. The pETDuet-tac-CBM2a was transformed into the host strain E. coli BL21 (DE3) to obtain recombinant cells containing surface-displayed CBM2a.

[0044] (2) single colony of Acetobacter xylinum ATCC in 50mL HS medium (glucose 40g / L, yeast extract 5g / L, peptone 5g / L, Na 2 HPO 4 2.7 g / L, citric acid 1.5 g / L) were statically cultured at 30°C for 2 day...

Embodiment 2

[0049] (1) Using PCR technology, a gene capable of displaying CBM that specifically binds to the cellulose crystal region on the surface of E. coli BL21 (DE3) was inserted into plasmid pETDuet-tac. It was digested with endonucleases NdeI and KpnI, and purified and recovered by PCR purification kit. Then, T4 ligase was used to ligate overnight at 16°C, and the gene capable of displaying CBM that specifically binds to the cellulose crystal region on the surface of E. coli E. coli BL21 (DE3) was ligated with the vector pETDuet-tac. The ligation product was transformed into competent cells E. coli DH5α, and the colony PCR and sequencing were verified to obtain the vector pETDuet-tac-CBM2a. The pETDuet-tac-CBM2a was transformed into the host strain E. coli BL21 (DE3) to obtain recombinant cells containing surface-displayed CBM2a.

[0050] (2) single colony of Acetobacter xylinum ATCC was placed in 50mL HS medium (glucose 40g / L, yeast extract 5g / L, peptone 5g / L, Na 2 HPO 4 2.7g / L...

Embodiment 3

[0053] (1) Using PCR technology, insert the gene SEQID NO.1 that can display CBM2a on the surface of Escherichia coli E.coli BL21 (DE3) into plasmid pETDuet-tac (using pETDuet as a template, and replacing the two T7 promoters on it with tac promoter, laboratory deposit). It was digested with endonucleases NdeI and KpnI, and purified and recovered by PCR purification kit. Then, T4 ligase was used to ligate overnight at 16°C, and SEQ ID NO. 1 was ligated with the vector pETDuet-tac. The ligation product was transformed into competent cells E.coli DH5α, and the colony PCR and sequencing were verified to obtain the vector pETDuet-tac-CBM2a. Then, using pETDuet-tac-CBM2a as a template, using NcoI and EcoRI restriction sites, and repeating the above restriction restriction steps, the green fluorescent protein gene of SEQ ID NO.2 was inserted into pETDuet-tac-CBM2a. The ligation product was transformed into competent cells E.coli DH5α, and the colony PCR and sequencing were verifie...

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Abstract

The invention discloses a bacterial cellulose-based biosensor, comprising bacterial cellulose and cells with surface displaying cellulose binding domain CBM, the cells are connected with bacterial cellulose through the cellulose binding domain CBM. The bacterial cellulose-based biosensor of the present invention can realize the efficient and specific immobilization of cells on the bacterial cellulose substrate, maintain the biological activity of the cells, enhance the output of fluorescent signals, provide enough pores for the detection objects to enter and exit, and significantly improve the detection performance. sensitivity.

Description

technical field [0001] The invention relates to the fields of molecular biology, genetic engineering, nanomaterials and biosensors, in particular to a bacterial cellulose-based biosensor and its application. Background technique [0002] Biosensors based on fluorescence detection play an important role in the fields of environmental pollutant detection, biochemical diagnosis, and biomedical sensing. So far, a variety of engineered strains have been publicly reported as sensors to detect chemicals including heavy metals, organic compounds, and antibiotics. However, the application of biosensors requires the immobilization of engineered strains on a material platform to maintain cell biological activity and enhance fluorescence signal output. In addition, the ideal biosensor platform also needs to have enough pores for the detection object to enter and exit, while minimizing the pollution to the environment. [0003] Bacterial cellulose (BC) refers to the synthesis of certai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/11C12N15/113C12N15/70C12N15/65G01N21/64C12R1/19
CPCC07K14/00C12N15/70C12N15/65G01N21/6428Y02A20/20
Inventor 龙凌凤孙付保胡芸谢乐
Owner JIANGNAN UNIV
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