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Method for regulating and controlling synthesis of EPS (extracellular polymeric substance) of clostridium acetobutylicum

A technology of Clostridium acetobutylicum and extracellular polymers, applied in the field of genetic engineering, can solve problems such as unclear mechanisms and achieve high regulatory effects

Active Publication Date: 2017-05-10
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as another form of EPS, the EPS formed by cells during immobilized fermentation has been rarely studied, especially the higher substrate tolerance and faster fermentation of EPS formed by immobilized cells. The mechanism of efficiency is still not very clear

Method used

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  • Method for regulating and controlling synthesis of EPS (extracellular polymeric substance) of clostridium acetobutylicum
  • Method for regulating and controlling synthesis of EPS (extracellular polymeric substance) of clostridium acetobutylicum
  • Method for regulating and controlling synthesis of EPS (extracellular polymeric substance) of clostridium acetobutylicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction method of Clostridium acetobutylicum CA_C2071 gene inactivation mutant.

[0030] 1. Design introns:

[0031] According to the CA_C2071 gene sequence (as shown in SEQ ID No: 1) of Clostridium acetobutylicum included in the NCBI database, a suitable insertion gene site (http: / / www.clostron.com) was designed with the help of software, and the insertion at Between 242-243 bases, an intron sequence is generated, and the sequence of the synthesized intron sequence S-242 is shown in SEQ ID NO:2.

[0032] 2. Construction of CA_C2071 insertion inactivation vector:

[0033] The vector PMTL007C-E2 was digested with XhoI and BsrGI, and its sequence is shown in SEQ ID NO:5. The digested product was purified by a purification kit (Takara), and connected to the intron sequence S-242 by one-step cloning (ClonExpress). Transform the recombinant plasmid connected by one-step cloning into Escherichia coli E.coli DH5a (our laboratory), spread it on LB plates cont...

Embodiment 2

[0040] Example 2: The process of immobilizing and fermenting the knockout bacteria and the starting bacteria in a 2L fermenter to synthesize extracellular polymeric substances (EPS).

[0041] Plate medium: yeast powder 3g / L, peptone 5g / L, glucose 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, phosphoric acid Dipotassium hydrogen 1g / L, ferrous sulfate heptahydrate 0.1g / L, agar 15g / L, the rest is water.

[0042] Seed medium: yeast powder 3g / L, peptone 5g / L, glucose 10g / L, ammonium acetate 2g / L, sodium chloride 3g / L, magnesium sulfate heptahydrate 3g / L, potassium dihydrogen phosphate 1g / L, phosphoric acid Dipotassium hydrogen 1g / L, ferrous sulfate heptahydrate 0.1g / L, the rest is water.

[0043] Fermentation medium: glucose 60g / L, ammonium acetate 2.2g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, sodium chloride 0.01g / L, magnesium sulfate heptahydrate 0.2g / L , ferr...

Embodiment 3

[0047] Example 3: Observing the structure and thickness of the biofilm of the knockout bacteria and the original bacteria using a laser confocal microscope.

[0048] Proteobacteria biofilms were cultured using the slide biofilm culture method, stained with fluorescein isothiocyanate (FITC)-labeled concanavalin A (FITC-ConA) and pyridium iodide (PI) double immunofluorescent technique, and laser Confocal microscopy (CLSM) was used to observe the biofilm structure and the distribution of exopolysaccharides in it, and the biofilm was scanned layer by layer, and the thickness of the biofilm was calculated according to the Z-axis distance of the three-dimensional coordinate system.

[0049] The specific operation is as follows:

[0050] (1) Preparation of biofilm specimens

[0051] Put a 25mm×75mm glass slide in a 100mL Schott bottle, and put 50mL fermentation broth into it for anaerobic static culture. After culturing for 6, 24 and 72 hours, the slides were taken out, washed repe...

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Abstract

The invention discloses a method for regulating and controlling synthesis of an extracellular polymeric substance of clostridium acetobutylicum. The method is characterized in that an intron sequence is interpolated into a gene Spo0A of the clostridium acetobutylicum by using a type II intron gene knockout technology. The invention provides a simple and efficient method for regulating and controlling EPS (Extracellular Polymeric Substance) synthesis; a recombinant strain obtained based on the method is relatively good in regulating and controlling effect on the EPS; in case of taking glucose as a carbon source, when cultivated in an anaerobic bottle with the volume of 100 ml by using a slide biomembrane cultivation method, a clostisdium acetobutylicum biomembrane is 5.81 mum in thickness, and the thickness of a strain biomembrane cultivated under the same condition is 24.6 mum.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for regulating the synthesis of Clostridium acetobutylicum extracellular polymer (EPS). Background technique [0002] Biofilm, also known as biofilm, is a dense and complex biological aggregate formed by microorganisms and their extracellular matrix (Extracellular polymeric substance, EPS) on the surface of biological or abiotic media. As the main component of biofilm, EPS plays an important role in maintaining the integrity of the structure and function of biofilm and smoothing the mass transfer channels of metabolites. EPS is an extracellular structure rich in macromolecular polysaccharides that cells independently synthesize under the induction of solid media. It has good water holding capacity, swelling property, and permeability. It can stabilize the hydration layer on the cell surface and pump out the reactants of the cell in time. , Create...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P7/26C12P7/16C12P1/04C12P19/04C12R1/145
CPCC12N15/74C12P1/04C12P7/16C12P7/26C12P19/04C07K14/33C12N2800/101C12R2001/145C12N1/205Y02E50/10
Inventor 应汉杰许佳慧柳东王岩岩沈晓宁刘俊
Owner NANJING UNIV OF TECH
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