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Method for electrotransformation of Clostridium thermocellum

A technology of Clostridium thermocellum and electrotransformation, which is applied in the field of microbial genetic engineering and can solve the problems of poor ethanol tolerance, low survival rate, low ethanol yield and the like

Inactive Publication Date: 2012-09-05
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0005] However, using Clostridium thermocellum to produce ethanol also has many technical difficulties, such as too low ethanol yield, poor ethanol tolerance, and many by-products. Genetic modification of Clostridium thermocellum for maximum input-output ratio looms
Unfortunately, there is no general and rapid method for genetic transformation of this microorganism, which limits the implementation of genetic engineering
JeffreyA.Sands once tried to transform the protoplasts of Clostridium thermocellum (Clostridium thermocellum), but its survival rate was low, only about 0.2% (Gottlund, Montenecourt et al.1988)
Lee R. Lynd laboratory successfully genetically modified Clostridium thermocellum (Tyurin, Desai et al.2004; Tyurin, Sullivan et al.2005; Olson, Tripathi et al.2010; Tripathi, Olson et al. 2010), however, the electroporation instrument used in the key electroporation process needs to be customized and has not yet been commercialized. Since most laboratories cannot be equipped with electroporation instruments with the same performance, the electroporation instrument of Clostridium thermocellum (Clostridium thermocellum) Transformation became the bottleneck for genetically engineering this strain

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Electrotransformation of Clostridium thermocellum ATCC27405 strain with pIKM1 plasmid.

[0024] 1. Inoculate the Clostridium thermocellum ATCC27405 strain frozen at -80°C into 4ml of preheated GS-2 medium at a ratio of 1 / 1000, and cultivate at 60°C until OD=0.4.

[0025] 2. Put the cultured seed solution into an anaerobic bottle containing 50ml GS-2 medium at a ratio of 1 / 1000.

[0026] 3. GS-2 medium according to the formula: KH 2 PO 4 1.5g, K 2 HPO 4 2.9g, Urea 2.1g, MgCl 2 .6H 2 O 1.0g, CaCl 2 .2H 2 O 150mg, FeSO 4 .6H 2 O 1.25mg, Cysteine ​​hydrochloride 1.0g, Resazurin 2.0g, Cellobiose 5.0g, Morpholinopropane sulfonic acid (MOPS) 10.0g, Yeast extract 6.0g, Sodium citrate. 2H 2 O 3.0g, add distilled water to make the volume to 1L.

[0027] 4. Cultivation temperature is 60°C, anaerobic, pH value is 7.4, cultured to OD=0.2, at this time, penicillin G sodium salt is added to make its final concentration 10μg / ml, and the culture is continued at 37°C for 2 hours.

[002...

Embodiment 2

[0036] Example 2: Plasmid pIKM1 electrotransformed Clostridium thermocellum ATCC27405 strain.

[0037] 1. Inoculate the Clostridium thermocellum ATCC27405 strain frozen at -80°C into 4ml of preheated GS-2 medium at 1 / 1000, and cultivate it at 60°C until OD=0.4.

[0038] 2. Put the cultured seed solution into an anaerobic bottle containing 50ml GS-2 medium at a ratio of 1 / 1000.

[0039] 3. GS-2 medium according to the formula: KH 2 PO 4 1.5g, K 2 HPO 4 2.9g, Urea 2.1g, MgCl 2 .6H 2 O 1.0g, CaCl 2 .2H 2 O 150mg, FeSO 4 .6H 2 O 1.25mg, Cysteine ​​hydrochloride 1.0g, Resazurin 2.0g, Cellobiose 5.0g, Morpholinopropane sulfonic acid (MOPS) 10.0g, Yeast extract 6.0g, Sodium citrate. 2H 2 O 3.0g, add distilled water to make the volume to 1L.

[0040] 4. Cultivation temperature is 60°C, anaerobic, pH value is 7.4, cultured to OD=0.3, at this time, penicillin G sodium salt is added to make its final concentration 30μg / ml, and the culture is continued at 45°C for 2 hours.

[0041] 5. In an anaerob...

Embodiment 3

[0049] Example 3: Plasmid pIKM1 electrotransformed Clostridium thermocellum ATCC27405 strain.

[0050] 1. Inoculate the Clostridium thermocellum ATCC27405 strain frozen at -80°C into 4ml of preheated GS-2 medium at 1 / 1000, and cultivate it at 60°C until OD=0.4.

[0051] 2. Put the cultured seed solution into an anaerobic bottle containing 50ml GS-2 medium at a ratio of 1 / 1000.

[0052] 3. GS-2 medium according to the formula: KH 2 PO 4 1.5g, K 2 HPO 4 2.9g, Urea 2.1g, MgCl 2 .6H 2 O 1.0g, CaCl 2 .2H 2 O 150mg, FeSO 4 .6H 2 O 1.25mg, Cysteine ​​hydrochloride 1.0g, Resazurin 2.0g, Cellobiose 5.0g, Morpholinopropane sulfonic acid (MOPS) 10.0g, Yeast extract 6.0g, Sodium citrate. 2H 2 O 3.0g, add distilled water to make the volume to 1L.

[0053] 4. The temperature is 60°C, anaerobic, pH value is 7.4, cultured to OD=0.4, at this time, penicillin G sodium salt is added to make its final concentration 50μg / ml, and the culture is continued at 60°C for 2 hours.

[0054] 5. In an anaerobic box, ...

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Abstract

The invention belongs to the field of microorganism gene engineering, and particularly relates to a method for electrotransformation of a thermophilic anaerobic cellulose-degrading bacterium of Clostridium thermocellum. The invention is characterized in that the method comprises the following steps: pretreating Clostridium thermocellum cells with an antibiotic for inhibiting bacterial cell wall synthesis (such as penicillin, ampicillin, etc.), and then transforming the Clostridium thermocellum cells by a common commercial electrotransformation machine so as to reach the purpose of introducing exogenous DNA into the Clostridium thermocellum. The method has the advantages that exogenous DNA is rapidly introduced into Clostridium thermocellum cells through a common commercial electrotransformation machine, which lays a foundation for the realization of bacterial gene engineering operations (such as gene overexpression, gene knockout, etc.), and the construction of gene engineering bacterial strains suitable for ethanol industrial production.

Description

Technical field [0001] The invention relates to the field of microbial genetic engineering, in particular to a method for electrotransforming Clostridium thermocellum (Clostridium thermocellum). Background technique [0002] With the continuous acceleration of economic and social development, the shortage of energy resources and environmental pollution have become increasingly prominent. Accelerating the development and utilization of biomass energy will play a positive role in alleviating the contradiction between national energy supply and demand, effectively protecting the ecological environment, and promoting sustainable social development. effect. The development of renewable energy resources and the protection of national energy security will become an important strategic task. The development and utilization of cellulosic ethanol is expected to solve the above problems (wu 2002; Cruz 2010; Service 2010). [0003] As one of the emerging energy sources, biomass energy is esti...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12R1/145
Inventor 崔球洪伟崔古贞刘亚君
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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