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Method for randomly inserting DNA fragment into bacillus subtilis chromosome and application thereof

A Bacillus subtilis, random insertion technology, applied in the high biological field, can solve the problems of false positives, inactivation of temperature-sensitive proteins, multi-copy insertion, etc., and achieve the effect of optimizing the expression level

Active Publication Date: 2021-06-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantage of this system is that it is screened at 50°C and there are multiple copy insertions; 50°C may inactivate temperature-sensitive proteins, and multiple copy insertions can lead to false positives

Method used

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  • Method for randomly inserting DNA fragment into bacillus subtilis chromosome and application thereof
  • Method for randomly inserting DNA fragment into bacillus subtilis chromosome and application thereof
  • Method for randomly inserting DNA fragment into bacillus subtilis chromosome and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Construction process of embodiment 1 plasmid pTMCZ and pUSIGH

[0024] Escherichia coli and Bacillus subtilis were cultured at 37°C with LB medium (peptone 1%, yeast powder 0.5%, sodium chloride 1%, pH 7.0). Transposon left repeat sequence ITR1 (Breton et al..2006.Appl EnvironMicrobiol.72:327-333.Doi:10.1128 / AEM.72.1.327-33.) and transcription terminator B0015 (http: / / parts .igem.org / Part:BBa_B0015) was entrusted to a biological company to synthesize, and then amplified by primers P1 / P2 to obtain the fragment ITR1-Transcription terminator. Using plasmid pNW33N (GenBank Number AY237122) as a template, primers P3 / P4 were used to amplify the chloramphenicol resistance gene. Using the plasmid pMarA (Breton et al..2006.Appl EnvironMicrobiol.72:327-333.Doi:10.1128 / AEM.72.1.327-33.) as a template, ITR2 was amplified using primers P5 / P6. Finally, using primers P1 / P6, ITR1-Transcription terminator, chloramphenicol resistance gene and ITR2 three fragments were connected by over...

Embodiment 2

[0026] Example 2 Random insertion of transposon into Bacillus subtilis chromosome

[0027] Such as figure 1 Plasmids pTMCZ and pUSIGH were co-transformed into B. subtilis 168 as indicated. The transformation of Bacillus subtilis was carried out with reference to the method established by Anagnostopoulos et al. (1961. J Bacteriol. 81:741-746.). After plasmid pTMCZ enters strain 168, fragment P zeo -ITR1-Transcription terminator-Cm-ITR2-Zeo will be inserted into the amyE site of the host chromosome through homologous recombination, and the plasmid pUSIGH can replicate independently after entering strain 168. Strain PDZC (pUSIGH) was obtained after resistance screening (10ppm chloramphenicol and 100ppm spectinomycin) and PCR verification. Culture strain PDZC(pUSIGH) to OD 600 After =1.0, add 1% xylose to induce the expression of the transposase gene Himar1 on the plasmid pUSIGH (induce 3 hours), and under the effect of transposase, the transposon ITR1-Transcription terminator...

Embodiment 3

[0028] Example 3 random insertion of target DNA into Bacillus subtilis chromosome

[0029] In order to use the transposon ITR1-Transcription terminator-Cm-ITR2 as a carrier, the target DNA is randomly inserted into the Bacillus subtilis chromosome, and the target DNA (such as figure 2 Middle EC) was inserted into the transposon by homologous recombination, and then xylose was used to induce transposon jumping (same as Example 2). In order to verify the feasibility, the expression cassette of methyl parathion hydrolase (MPH) P NBP3510 -mpd(Zhou et al..2019.Microb Cell Fact.18(1):111.Doi:10.1186 / s12934-019-1159-0) was inserted into the transposon ITR1-Transcription terminator-Cm-ITR2, and a strain was generated PDZCM (pUSIGH). The specific steps are: using the total DNA of the strain PDZC (pUSIGH) as a template, using primer pairs P20 / P21 and P22 / P23 to amplify LF-ITR1-Transcription terminator and ITR2-RF respectively; using the strain PD8NM (Zhou et al..2019.Microb Cell Fac...

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Abstract

The invention discloses a method for randomly inserting a DNA fragment into a bacillus subtilis chromosome and application of the method. The invention relates to a composition, which consists of a transposon element or a plasmid containing the transposon element and a plasmid pUSIGH. The composition is applied to randomly inserting the target DNA fragment into the bacillus subtilis chromosome. After the composition is co-transformed into bacillus subtilis, the transposon element or a plasmid containing the transposon element can integrate the transposon element into a host chromosome through homologous recombination, and pUSIGH can be normally and independently copied in a host. Transposase gene expression on pUSIGH is induced by using xylose, and the transposon is randomly inserted into the chromosome under the action of transposase. Then, clones with relatively high target protein expression level can be screened out.

Description

technical field [0001] The invention belongs to the field of biological high technology, and discloses a method for randomly inserting DNA fragments into the chromosome of bacillus subtilis and an application thereof. Background technique [0002] Bacillus subtilis is an important recombinant protein expression host, which has the advantages of good safety, low fermentation cost, strong protein secretion ability and rapid growth. When constructing an expression strain, a multi-copy plasmid can be used to carry the target gene, or the target gene can be integrated into the host chromosome. These two ways are called plasmid expression and chromosomal integration expression respectively. The disadvantage of plasmid expression is poor stability and usually carries resistance genes, and the advantage is high gene copy number. The chromosomal integration type has good expression stability and no resistance gene problem, but the disadvantage of this method is that the number of g...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N15/67C12N1/21C12N15/55C12R1/125
CPCC12N15/75C12N15/67C12N9/16C12Y301/08001C12N2800/90Y02A50/30
Inventor 闫新叶斌成明根
Owner NANJING AGRICULTURAL UNIVERSITY
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