Method for achieving gene edition screening based on LAC4 gene

A gene editing and gene technology, applied in the field of gene editing and screening of Kluyveromyces lactis, can solve the problems of cumbersome screening and identification of positive strains, achieve the effect of improving the positive rate of screening, eliminating the need for PCR screening, and saving time and cost

Active Publication Date: 2020-06-05
KANGMA SHANGHAI BIOTECH LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It mainly solves the technical problem that the CRISPR / Cas9 system is used as a gene editing tool in the prior art, and the screening and identification of positive strains is relatively cumbersome

Method used

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  • Method for achieving gene edition screening based on LAC4 gene
  • Method for achieving gene edition screening based on LAC4 gene
  • Method for achieving gene edition screening based on LAC4 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Verification of the growth of LAC4 knockout strains on SC-lactose plates

[0056] 1.1 Preparation of yeast medium SC-lactose with lactose as the sole carbon source

[0057] Weigh 1.58g of sugar-free SC medium (synthetic medium, without carbon source) powder, dissolve it in 180mL of water, adjust the pH to 5.6-6.4, add 3g of agarose, and autoclave at 115°C for 30min. Dissolve 4g of lactose in 20mL of water, filter and sterilize with a 0.2μm filter membrane for later use. Add 20mL of the above-mentioned 2% lactose solution sterilized by filtration to the SC medium after autoclaving, mix well, and put it into a sterile petri dish when it is cooled to about 60°C to prepare SC-lactose solid medium .

[0058] 1.2 Observe the growth difference between the LAC4 gene knockout strain and the wild-type strain on SC-lactose medium

[0059] Take the wild-type K. lactis strain and the ΔLAC4 strain (the genotype is the entire coding region sequence of the LAC4 gene that ...

Embodiment 2

[0060] Example 2: Mock Screening of LAC4 Gene Knockout Strains

[0061] The wild-type K. lactis strain adjusted to approximately the same OD600 value (about 5.0) was mixed with the ΔLAC4 strain at a ratio of 10:1 (the mixed bacterial liquid was the transformation liquid simulated when the LAC4 gene was knocked out on the K. lactis strain), 10 6 After dilution, 30 μL was spread on a YPD plate and incubated at 30°C.

[0062] After the colonies grow, pick 66 single colonies and spot them on the YPD plate (plate A) and the SC-lactose plate (plate B) at the same time, so that each single colony is in a one-to-one correspondence between plate A and plate B . After about a day, the colonies on plate A and plate B grew as follows figure 2 As shown, the left picture is plate A, and the right picture is plate B. according to figure 2 It can be found that the diameters of the colonies grown on plate A are basically the same, while the diameters of some colonies on plate B are obvio...

Embodiment 3

[0064] Example 3: Simulation screening of bacterial strains containing the LAC4 gene

[0065] Mix the wild-type K. lactis strain adjusted to approximately the same OD600 value (about 5.0) and the ΔLAC4 strain at a ratio of 1:100 (the mixed bacterial solution is the transformation solution for simulating the restoration of the LAC4 gene on the ΔLAC4 strain), and take 30 μL to spread Culture on SC-lactose plate at 30°C. After culturing for about 16 hours, only 4 colonies grew on the SC-lactose plate, as Figure 4 Shown is the growth of wild-type strain Y1140 and ΔLAC4 strain 1:100 mixed on SC-lactose medium. The 4 single clones were picked and identified by colony PCR. Identification results such as Figure 5 As shown, the negative band is 2741bp, and the positive band is 8438bp, according to Figure 5 The results of electrophoresis showed that all the four single clones selected were strains containing the LAC4 gene, and the screening positive rate was 100%.

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Abstract

The invention discloses a method for achieving gene edition screening based on an LAC4 gene. The method comprises the following steps: 1, performing gene transformation, namely performing gene transformation so as to disable an LAC4 gene on an original strain chromosome to express an LAC4 protein with normal functions so as to obtain a strain with LAC4 gene defect, wherein the strain with the LAC4gene defect cannot grow normally on a culture medium with lactose as a sole carbon source; 2, performing gene recovery, namely connecting a target gene with a normal LAC4 gene to form a donor fragment, and transferring the donor fragment into the strain with the LAC4 gene defect so as to recover normal expression functions of the LAC4 gene; and 3, culturing the strain after gene recovery on the culture medium with lactose as the sole carbon source, wherein a strain which can grow normally is a strain after recovery of the LAC4 gene and is also a strain that the target gene is inserted normally. Compared with a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / Cas9) system without a screening function, the system is capable of rejecting most strains with negative geneedition through screening, the screening positive rate can be greatly increased, PCR (polymerase chain reaction) is barely needed, and the time and cost can be saved.

Description

technical field [0001] The invention belongs to the technical field of Kluyveromyces lactis gene editing and screening, and in particular relates to a method for realizing gene editing and screening based on the LAC4 gene. Background technique [0002] Kluyveromyces lactis (hereinafter referred to as K.lactis) is a yeast widely used in industry. Compared with other yeasts, it has many advantages, such as super strong secretion ability, good large-scale fermentation characteristics, high Food safety level and the ability to modify proteins after translation. Therefore, how to improve the protein expression ability of K. lactis has become the focus of research, and gene editing of strain chromosomes is one of the powerful means. [0003] The CRISPR / Cas9 system is a powerful gene editing tool developed in recent years, but its editing efficiency in K. lactis is not very high, which brings inconvenience to the screening and identification of positive strains. The effect of dir...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/90C12N1/19C12R1/645
CPCC12N15/905C07K14/39C12N2800/80C12N2810/10
Inventor郭敏姜灵轩李海洋占魁娄旭邓蜜妮范万巧杨旭许乃庆于雪
OwnerKANGMA SHANGHAI BIOTECH LTD