Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene

A Bacillus subtilis, resistance marker technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc. The effect of high efficiency, simple construction of integrated mutation vector, and simple process

Inactive Publication Date: 2011-04-20
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above method can achieve the purpose of screening without antibiotic resistance markers, the

Method used

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  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene
  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene
  • Antibiotic resistance maker-free bacillus subtilis constructing method and method for screening bacillus subtilis with inactivated target gene

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Embodiment 1B

[0048] Construction of embodiment 1BS-PS mutant strain

[0049] (1) Construction of SpovAF-CM-LysA homologous exchange fragment

[0050] build route see figure 2 . With spoVAF-F (SEQ ID NO.1) and spoVAF-R (SEQ ID NO.2) as primers, with Bacillus subtilis 168 (BS168) genomic DNA as template PCR amplification spoVAF gene; with LysA-F (SEQ ID NO. NO.3) and LysA-R (SEQ ID NO.4) were primers, and the Lys gene was amplified by PCR using BS168 genomic DNA as a template; CM-F (SEQ ID NO.5) and CM-R (SEQ ID NO. 6) was used as a primer, and the CM (chloramphenicol resistance gene) including the promoter and the terminator was amplified by PCR using the pJC164.3 plasmid (BGSC, containing the CM gene) as the template. The three amplified genes were respectively cloned into the pMD19-T (purchased from Takara Company) vector, and after verification and correct sequencing, they were named pMD19-T-spoVAF, pMD19-T-LysA, and pMD19-T-CM respectively. Store at -20°C for later use.

[0051] T...

Embodiment 2

[0064] Example 2 Utilize the non-antibiotic resistance marker gene screening technology to screen the Bacillus subtilis with target gene inactivation

[0065] (1) Construction of BS-PS strain protease nprE and aprE inactivation integration vector

[0066] In the present invention, two protease genes aprE and nprE secreted by Bacillus subtilis are selected, and an insertion inactivation strategy is adopted to conduct mutation inactivation research. Using E. coli pMD19-T as the backbone, a general integration vector PLC-T was constructed. The PLC-IaprE-T integration vector for insertion inactivation of aprE gene and the PLC-InprE-T integration vector for insertion inactivation of nprE gene were constructed on the basis of general vectors. Build routes like Figure 5 shown.

[0067] The specific construction process of the PLC-T integration vector is as follows:

[0068]Using P43-F (SEQ ID NO.9) / P43-R (SEQ ID NO.10) as primers, using BS168 genomic DNA as a template for PCR am...

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Abstract

The invention discloses an antibiotic resistance maker-free bacillus subtilis constructing method and a method for screening bacillus subtilis with an inactivated target gene. The method comprises the steps of: constructing two homologous exchange segments by using bacillus subtilis 168 as a starting strain; constructing a controllable lysine auxotroph strain BS-PS by two dual exchange processes on the basis of a homologous recombination principle; constructing a universal integrated mutation carrier PLC by using a colibacillus clone carrier pMD19-T as a framework; and constructing an integrated carrier PLC-InprE of the insertional inactivated nprE by using the neutral protease nprE of the bacillus subtilis as the inactivated target gene and by adopting an insertional inactivation strategy based on a programmable logic controller (PLC), and converting the PLC-nprE into BS-PS, controlling lysine to synthesize a screening marker by a single exchange process and a spontaneous single exchange process and screening protease insertional inactivation mutant strains by combining the result of polymerase chain reaction (PCR) verification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for constructing Bacillus subtilis without antibiotic resistance markers and a method for screening Bacillus subtilis with inactivated target genes. Background technique [0002] Bacillus subtilis is recognized as a safe strain (GRAS), which can secrete and express active products from exogenous genes. In addition, Bacillus subtilis has a clear genetic background, rapid growth, simple culture conditions, good fermentation foundation and production technology, making it widely used in scientific research and industrial and agricultural production. As an important industrial microorganism, B. subtilis has the advantage of being developed as a food-grade expression host, reducing the cost of downstream isolation and purification of recombinant biological products used in the food industry. [0003] At present, the biggest problem of B. subtilis as a host is the protease secreted...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/10C12N15/63C12Q1/68C12Q1/04
Inventor 陆兆新张充别小妹吕凤霞王煜赵海珍
Owner NANJING AGRICULTURAL UNIVERSITY
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