High-throughput pichia pastoris screening method based on droplet micro-fluidic chip

A microfluidic chip, Pichia pastoris technology, applied in the field of microbial biology

Active Publication Date: 2020-09-29
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There is no report on the droplet microfluidic high-throughput screening of Pichia pastoris

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Effect test

Embodiment 1

[0030] Example 1 Droplet microfluidic high-throughput screening method for high-yield xylan of Pichia pastoris

[0031] 1. Construction and expression of Pichia pastoris strain with xylanase fusion green fluorescent protein

[0032] The fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene was amplified by PCR, and cloned into the expression vector pPIC9K of Pichia pastoris to construct the plasmid pPIC9K-xyn5-gfp of xylanase fusion green fluorescent protein, using EcoRI It was verified by double enzyme digestion with NotⅠ. From Figure 2(a), two bands of xyn5-gfp fusion fragment and pPIC9K vector fragment can be seen; the plasmid was electrotransformed into Pichia pastoris GS115, and the genome was extracted for PCR amplification. To increase the integration efficiency, the results of agarose gel electrophoresis in Figure 2(b) show that the amplified band is consistent with the size of the exogenous gene fragment xyn5-gfp, and the recombinant Pichia past...

Embodiment 2

[0051] Droplet microfluidic high-throughput screening method for high-production cellulase from Pichia pastoris

[0052] Cellulase-producing Pichia strain GS115 / pPIC9K-cbh1 was constructed using the cbh1 gene derived from Penicilliuoxalicum, and the strain was treated according to steps 2-4 in Example 1, wherein the strain was mutagenized by ARTP to establish a mutant library, the mutagenesis time was 60s, and the strains in the Pichia mutant library were sonicated for 1min (the total power of the instrument was 950W, the power used was 4%, the ultrasound was 9.9s, and the stop was 9.9s, a total of 1min), and then embedded in OD 600 =0.3 for single droplet embedding and droplet culture, at 30°C, after 40 hours of incubation, detect the fluorescence signal intensity of the cellulase substrate in the droplet for microfluidic sorting of the droplet, and screen out the one with higher signal intensity or Several liquid droplets were subjected to shake-flask fermentation to screen ...

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Abstract

The invention belongs to the technical field of microbial biology, and relates to a high-throughput pichia pastoris screening method based on a droplet micro-fluidic chip. The screening method comprises the following steps: step 1, constructing pichia pastoris; step 2, constructing a pichia pastoris mutant library; step 3, carrying out single-droplet embedding and droplet culture on strains in thePichia pastoris mutant library to obtain droplets containing Pichia pastoris mutants; step 4, carrying out droplet microfluidic sorting according to the intensity of fluorescence in the droplets, andscreening out the droplets; and 5, respectively carrying out shake-flask fermentation and re-screening on the droplets to obtain a pichia pastoris strain with improved xylanase secretion capacity. The pichia pastoris strain of xylanase fused fluorescent protein is screened by adopting a droplet microfluidic technology, and the screening flux can reach 100 thousand strains per hour; by using the method, for screening of a million-level strain library, only 10 hours are needed, and the total volume of consumed fluorescent reagents is 100 microliters.

Description

technical field [0001] The invention belongs to the field of microbial biotechnology. More specifically, the present invention relates to a high-throughput screening method for Pichia pastoris based on a droplet microfluidic chip. Background technique [0002] As a mature exogenous protein expression system, Pichia pastoris has realized the expression of more than 1,000 exogenous proteins, and its excellent expression characteristics have been widely verified, such as high secretion efficiency, simple genetic operation, and low culture cost Wait. At present, for the high-efficiency expression of foreign proteins in Pichia pastoris, on the one hand, the modification of foreign genes at the molecular level can greatly improve the expression of foreign genes; on the other hand, high-throughput screening techniques are used to obtain high-expression strains . In the past, the screening methods for Pichia pastoris were mainly based on traditional methods such as flat plates an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N13/00C12N1/04C12R1/84
CPCC12N9/248C12N13/00C12Q1/04C07K2319/60G01N2333/39
Inventor 涂然王钦宏吕彤
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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