High-conversion-rate bacillus subtilis and structuring method thereof

A Bacillus subtilis and gene technology, applied in the field of microorganisms, can solve the problems of low transformation efficiency of wild-type Bacillus subtilis, and achieve the effects of large application potential, easy operation and elimination of resistance.

Inactive Publication Date: 2016-08-17
SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to overcome the problem of low transformation efficiency of wild type Bacillus subtilis at present, the object of the present invention is to provide a kind of high transformation rate Bacillus subtilis and its preparation and application

Method used

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  • High-conversion-rate bacillus subtilis and structuring method thereof
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  • High-conversion-rate bacillus subtilis and structuring method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of expression plasmid pMK4-cre, expression plasmid pMK4-comk and recombinant plasmid pGSNE-nprlR-comk

[0045] 1. Construction of pMK4-cre

[0046] pMK4-cre is based on the pMK4 vector, which is connected with a Cre enzyme expression gene cre containing a Pspac promoter, and is used for expressing Cre enzyme in Bacillus picaridis. The sequence of pMK4-cre is shown in SEQ NO.1, specifically:

[0047]

[0048] The Pspac-cre fragment was amplified by PCR using the plasmid pDGC as a template, using Pcre-F (SEQ ID NO.5) as an upstream primer, and Pcre-R (SEQ ID NO.6) as a downstream primer.

[0049] Pcre-F (SEQ ID NO.5), specifically: taccgaattctacacagcccagtccaga.

[0050] Pcre-R (SEQ ID NO.6), specifically: ctaggaattcgcatgcctaatcgccatcttccag.

[0051] The PCR reaction system is as follows:

[0052]

[0053] Run the following programs on a Mastercycler ordinary PCR machine (Eppendorf):

[0054] (1) 98°C, 2min; (2) 98°C, 15sec; (3) 55°C, 15sec; (...

example 2

[0084] Example 2 Construction of Bacillus subtilis 164S

[0085] Using plasmids pMK4-comk, pGSNE-nprlR-comk, and pMK4-cre to transform Bacillus subtilis A164 (the DDBJ / EMBL / GenBank access number of A164 genome sequence is no.CP011115), and finally obtained an easy-to-operate strain of subtilis with high transformation efficiency Bacillus 164S, see flow chart Figure 4 .

[0086] The specific operation is as follows:

[0087] First, pMK4-comk was transformed into Bacillus subtilis A164. The transformation adopts the method of hyperosmotic electric shock transformation,

[0088] a) inoculate Bacillus subtilis A164 in 3mL LB medium, cultivate overnight;

[0089] b) Transfer 2.6mL of overnight culture solution to 40mL (LB+0.5M sorbitol) and culture at 37°C and 200rpm until OD600=0.8;

[0090] c) Bath the bacterial solution in ice water for 10 minutes, then centrifuge at 5000g for 5 minutes at 4°C to collect the bacterial cells;

[0091] d) Use 30mL pre-cooled electroporation...

example 3

[0107] Example 3 Knockout of Bacillus subtilis 164S amylase gene

[0108] The gene manipulation of Bacillus subtilis 164S can be conveniently carried out by adopting the method of inducing transformation. After using the restriction endonuclease Kpn I to cut the amylase knockout plasmid pDG364, without cleaning the product, directly transform into Bacillus subtilis 164S:

[0109] a) Inoculate the plate-activated Bacillus subtilis 164S colony into a 3mL LB test tube, and incubate at 37°C and 200rpm for 10-12h;

[0110] b) Transfer 300 μL culture solution to a preheated 3 mL LB test tube, add filter-sterilized xylose solution to a final xylose concentration of 1%, and incubate at 37°C and 200 rpm for 3 hours;

[0111] c) Dispense into 2mL Eppendorf tubes, add linearized plasmid at a ratio of 100ng DNA / 100μL culture solution, and incubate at 37°C and 200rpm for 1.5h. Coat the chloramphenicol resistant plate with 5 µg / mL. Incubate overnight at 37°C.

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Abstract

The invention belongs to the technical field of microorganisms and particularly relates to high-conversion-rate bacillus subtilis and preparation and application thereof. The problem that the bacillus subtilis A164 is low in conversion efficiency is solved through a plasmid expression method, then a linear fragment containing a comk expression cassette is integrated on an A164 genome through an induced conversion method, and resistance is eliminated by the aid of a cre/lox specific recombination system to obtain bacillus subtilis A164S which is free of resistance marker, easy to operate and high in conversion efficiency and secretion capability. Research results show that conversion efficiency of the bacillus subtilis A164S is more than 1000 times of that of the bacillus subtilis A164; the bacillus subtilis A164S can effectively take in homologous DNA, can efficiently secrete heterologous proteins, and has high application potential in recombinant expression of exogenous genes.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a high-transformation bacillus subtilis and its preparation and application. Background technique [0002] Bacillus subtilis is recognized as a generally recognized as safe (GRAS, Generally Recognized as Safe) organism. Because of its non-pyrogenic, high secretion, easy to cultivate, and its products can be directly used in people's daily use, it has become an industrial polypeptide product. It is the first choice for secreted expression of pharmaceuticals and enzyme preparations. Compared with the model strain A168, Bacillus subtilis A164 (ATCC 6051a) has excellent biochemical characteristics such as growth characteristics and secretion ability, and is an ideal starting strain for the construction of fermentation industry production bacteria. However, due to the extremely low DNA transformation efficiency of A164 and the difficulty of gene manipulation, the w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12R1/125
CPCC07K14/32C12N9/54C12N15/75C12N2800/101
Inventor 孙俊松纪明华史吉平姜标
Owner SHANGHAI ADVANCED RES INST CHINESE ACADEMY OF SCI
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