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A high-throughput screening method for membrane imprinting of genetically engineered bacteria secreting lipase

A technology of genetically engineered bacteria and lipase genes is applied in the field of bioengineering to achieve the effects of cost saving, wide application range, and sensitive and accurate results

Active Publication Date: 2018-02-13
GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has improved the screening efficiency, these methods have certain limitations in ensuring the integrity of the subcloning library and screening under special conditions (such as high methanol concentration, high pH, ​​etc.)

Method used

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  • A high-throughput screening method for membrane imprinting of genetically engineered bacteria secreting lipase
  • A high-throughput screening method for membrane imprinting of genetically engineered bacteria secreting lipase
  • A high-throughput screening method for membrane imprinting of genetically engineered bacteria secreting lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Screening of lipase genetically engineered bacteria with different enzyme-producing activities

[0044]The Rhizomucor miehei lipase gene rml and the pPIC9K plasmid were digested with EcoR I and Not I at the same time, connected with T4 ligase, transformed into E. coli DH5α competent cells, and the transformed cells were coated with 100ug / mL ampicillin On the LB plate of penicillin, culture upside down at 37°C for 12-16 hours, pick 2-5 single colonies from the plate, inoculate in LB liquid medium, shake overnight at 37°C and 250r / min. A small amount of plasmid DNA was extracted with a plasmid kit, identified by double enzyme digestion with EcoRI and Not I, and the successfully ligated pPIC9K-rml plasmid was obtained. The pPIC9K-rml plasmid was linearized with SalI and electrotransformed into the host Pichia pastoris GS115, spread on the MD plate, and cultured at 28°C for 2-3 days to obtain the lipase gene of Rhizomucor miehei with different enzyme production a...

Embodiment 2

[0048] Example 2: High-throughput screening of lipase genetically engineered bacteria mutant library

[0049] The Rhizomucor miehei lipase gene is transformed by the error-prone PCR method and transformed into Pichia pastoris X33. The transformation method is the same as described in Example 1, and the transformed Pichia pastoris X33 is inoculated on a solid containing YPD. The medium has a diameter of 9 mm on a petri dish, and cultured at 30° C. to obtain a mutant library of Rhizomucor miehei lipase Pichia pastoris genetically engineered bacteria. Place a cellulose acetate membrane with a diameter of 9mm flat on the YPD solid medium, press lightly with a coating rod to completely wet the filter paper membrane and drive out air bubbles, transfer all the colonies to the cellulose acetate membrane, and culture the YPD solid Store the base plate at 4°C; place a nylon membrane of the same size with a pore size of 0.45 μm on another BMMY solid medium plate with 1% methanol added to...

Embodiment 3

[0052] Example 3: High-throughput screening of high-temperature-resistant lipase genetically engineered bacteria

[0053] The Rhizomucor miehei lipase gene is transformed and transformed into Pichia pastoris GS115 by the method of error-prone PCR. The transformation method is the same as that described in Example 1, and the transformed Pichia pastoris GS115 is inoculated on the solid containing MD. The medium has a diameter of 9 mm on a petri dish, and cultured at 30° C. to obtain a mutant library of Rhizomucor miehei lipase Pichia pastoris genetically engineered bacteria. Place the filter paper membrane with a diameter of 9mm flat on the MD solid medium, press lightly with a coating rod to completely wet the filter paper membrane and drive away the air bubbles, so that all the colonies are transferred to the PVDF membrane, and place the MD solid medium plate on Store at 4°C; place a PVDF membrane of the same size with a pore size of 0.22 μm on another BMMY solid medium plate ...

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Abstract

The invention discloses a membrane blotting high-throughput screening method for secreted lipase gene engineering bacteria. According to the method, screened strains are inoculated to a blotting membrane by a load membrane for induced culture, secreted lipase is bonded to the blotting membrane, and the secreted lipase gene engineering bacteria are subjected to high-throughput screening by using the principle that a lipase catalyzed ester hydrolysis product can develop a color through a color developing agent. By adopting the method, the secreted lipase gene engineering bacteria with special performance, particularly lipase gene engineering strains with high temperature resistance, methanol resistance and the like, can be screened. The method can be used for high-throughput screening of massive lipase gene engineering bacteria mutant libraries, and can obtain high screening efficiency and ensure the integrity of a sub-clone library to realize screening of the sub-clone library under multiple conditions, so the method is wide in application range, simple and convenient in operation and accurate in result.

Description

Technical field: [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-throughput screening method for membrane imprinting of secreted lipase genetically engineered bacteria. Background technique: [0002] Lipase is one of the important industrial enzyme preparations, which can catalyze lipolysis, transesterification, ester synthesis and other reactions, and is widely used in food industry, paper industry, leather industry, feed industry, pharmaceutical catalytic synthesis, oil cheese processing and biodiesel production and other industries. However, lipase itself is a kind of protein, which can only play its catalytic role under certain conditions. Once the external conditions such as temperature, pH and organic solvent concentration are changed, it will be easily inactivated. In order to obtain lipases with higher enzymatic activity and more stable performance, researchers usually use methods such as molecular evolution...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/44C12Q1/04C12N15/81C12N15/63
Inventor 吕鹏梅何东王治元袁振宏罗文刘姝娜李志兵杨改秀杨玲梅
Owner GUANGZHOU INST OF ENERGY CONVERSION - CHINESE ACAD OF SCI
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