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Bacillus subtilis, biofilm and construction and application thereof

A Bacillus subtilis, biofilm technology, applied in the field of synthetic biology, can solve problems such as hindering the application of living biofilm functionalized materials and hidden dangers of Escherichia coli biosafety, achieve increased chemical and thermal stability, and achieve large-scale production. Effect

Inactive Publication Date: 2017-05-31
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitations of its own secretion system (especially the limitation of the double membrane), the use of biofilms to display fusion peptides cannot exceed 50 amino acids, and the biological safety hazards of Escherichia coli have greatly hindered the development of living biofilm functional materials. application

Method used

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  • Bacillus subtilis, biofilm and construction and application thereof
  • Bacillus subtilis, biofilm and construction and application thereof
  • Bacillus subtilis, biofilm and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1: Construction of the Bacillus subtilis mutant strain carrying tasA-R expression plasmid

[0083] (1) Construction of Bacillus subtilis mutant strain:

[0084] The main components of the biofilm of Bacillus subtilis are exopolysaccharides and amyloid, which are mainly encoded by the eps gene cluster and the tasA gene.

[0085] The amyloid-encoding gene tasA of the original Bacillus subtilis 3610 that produced the biofilm and the gene sinR encoding the biofilm inhibitor were knocked out on the genome to construct the Bacillus subtilis mutant strain ΔtasAΔsinR001; The amyloid-encoding gene tasA, the exopolysaccharide-producing gene cluster epsA-O and the biofilm inhibitor-encoding gene sinR were knocked out on the genome to construct the Bacillus subtilis mutant strain ΔtasAΔsinRΔeps002; the strain was released in June 2016 On the 6th, it was stored in the China General Microorganism Culture Collection Center (CGMCC), (CGMCC number: 12600).

[0086] Knock ou...

Embodiment 2

[0167] Embodiment 2: Construction of the Bacillus subtilis mutant strain carrying tasA-R expression plasmid

[0168] In addition to the plasmid expression method described in Example 1, a direct genome replacement protocol for B. subtilis can also be performed. Instead of knocking out biofilm-encoding genes, tasA was directly replaced by tasA-R genes at the genome level.

[0169] Directly integrate and express the fusion tasA-R biofilm at the genome level, and there are two ways of constitutive expression and promoter inducible expression.

[0170] 1. Construction of constitutive tasA-R biofilm expression genome integration strain:

[0171] Taking the integration of tasA-histag, tasA-mefp3-histag, tasA-mefp5-histag, tasA-mcherry-histag and tasA-oph into the genome as an example, the following steps are included:

[0172] (1) Using the expression vector in Example 1 as a template, design specific upstream and downstream primers to expand the tasA-histag, tasA-mefp3-histag, ta...

Embodiment 3

[0230] Example 3: Biomembrane platform comprising tasA-Histag fusion protein for biocatalysis

[0231] The strain expressing tasA-histag biofilm can be a constitutive expression of genome integration, or an IPTG-inducible expression or a strain carrying an IPTG-induced expression plasmid. Here, the (002) mutant strain of the triple mutation of ΔtasAΔsinRΔeps in Example 1 Carry pHT01-tasA-histag expression plasmid, IPTG induces TasA-Histag as an example, the specific steps are:

[0232] 1. Transformation: the expression plasmid is transformed into a biofilm mutant strain (002), and the transformation method is as follows:

[0233] (1) Activate the (002) mutant strain of the ΔtasAΔsinRΔeps triple mutation in Example 1 to be transformed.

[0234] (2) Pick a fresh single clone and inoculate it in 3ml of 2xYT medium, culture it overnight at 37°C with shaking.

[0235] (3) Inoculate the supernatant bacteria in 5ml of medium A at a ratio of 1:100, and culture with shaking at 37°C f...

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Abstract

The invention provides bacillus subtilis, a biofilm and construction and application thereof. A biofilm exhibition platform is a modified biofilm of the bacillus subtilis, wherein a TasA protein in the bacillus subtilis biofilm is modified into a TasA-R protein, is exhibited on the biofilm and has functionality. Gene modification and regulation and control of a main protein of the bacillus subtilis biofilm, namely a TasA starch sample protein, are carried out, and the exhibition platform of the bacillus subtilis biofilm is realized; compared with previous reports, the platform has the advantages of higher secretion capability, more complete functionality, higher biological safety, capability of being regulated and controlled and the like; the exhibition technology of the bacillus subtilis biofilm is widely applied.

Description

technical field [0001] The invention relates to the fields of synthetic biology, genetic engineering technology and biological materials, in particular to a bacillus subtilis, a biofilm produced by the bacillus subtilis and a construction method and application thereof. Background technique [0002] Biofilm is a complex composed of microorganisms, polysaccharides, DNA, proteins and lipids. Biofilm is usually associated with infection and disease of pathogenic bacteria. The biofilm secreted by many bacteria is the culprit of infection and causes drug resistance. and difficult to remove. Streptococcus sanguis was injected into the rabbit body through the catheter, and within 30 minutes, it could be adsorbed on the surface of the sterilized catheter, and then streptococcal colonies formed near the thrombus, and were observed to be surrounded by fibrosis capsules, which can be seen Effect of streptococcal biofilms on infection and prevention of leukocytes. The same cystic fibr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC12N15/75C07K14/32C12N2800/101
Inventor 钟超黄娇芳张琛刘苏莹
Owner SHANGHAI TECH UNIV
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