Traceless modification method of bacillus subtilis genome

A Bacillus subtilis, modification method technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria and other directions, can solve the problems of high false positive rate, difficulty in screening positive recombinant strains, low absorption capacity, etc. The effect of chemical preparation process and excellent ability to transform exogenous DNA

Active Publication Date: 2013-12-18
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some defects in this method: on the one hand, Bacillus subtilis competent cells have a low ability to absorb exogenous DNA (such as the Spizizen method), resulting in low efficiency of the first gene recombination of exogenous DNA; on the other hand, the entire genome modification The second intramolecular gene recombination involved in the process is only negative screening, and the false positive rate is high, which brings certain difficulties to strain screening; meanwhile, the efficiency of the second intramolecular homologous recombination is low, and the literature report is only 10 -7 ~10 -6 It is also difficult to screen out positive recombinant strains

Method used

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  • Traceless modification method of bacillus subtilis genome
  • Traceless modification method of bacillus subtilis genome
  • Traceless modification method of bacillus subtilis genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Construction of the template plasmid pSS containing the I-SceI restriction site (see figure 1 A)

[0043] Use the PCR reaction to use the pC194 plasmid as a template to obtain the cat gene using the upstream and downstream primers pSS-P1 and pSS-P2, and use the B. subtilis168 genome as a template to obtain the upp gene using the upstream and downstream primers pSS-P3 and pSS-P4, and then use the above two A fragment was used as a template, and the recombinant fragment cat-upp was obtained by fusion PCR reaction using upstream and downstream primers pSS-P1 and pSS-P4. The recombinant fragment and the pUC18 (universal plasmid) plasmid were subjected to enzyme digestion, enzyme ligation, transformation, verification, etc., to obtain the basic operation plasmid pSS (see Table 2 for the primer sequences used).

[0044] 2. Construction of temperature-sensitive expression plasmid pEBS-copl (see map figure 1 b)

[0045] The erm gene was obtained by using the pE194 plasmid...

Embodiment 2

[0061] Example 2: Gene mutation introduced by traceless genetic manipulation (ccpN*G130T) see image 3

[0062](1) Using the plasmid pSS as a template, primers ccpN-Mut-P3 and ccpN-Mut-P4 as amplification primers, and PCR to obtain positive and negative screening boxes; using the B. subtilis168 genome as a template, using ccpN-Mut-P1 and containing The target gene mutation (G130T) ccpN-Mut-P2 was used as a primer, and the upstream homology arm F-1 (1379bp) containing the target mutation (G130T) DR sequence was obtained by PCR; B. subtilis168 was used as a template to contain the target gene mutation ( ccpN-Mut-P5 and ccpN-Mut-P6 of G130T) were used as primers, and the downstream homology arm B-1 (1339bp) containing the DR sequence of the target mutation (G130T) was obtained by PCR; the DR containing the target mutation (G130T) The upstream homology arm F-1 of the sequence, the positive and negative screening box and the downstream homology arm B-1 containing the DR sequence o...

Embodiment 3

[0068] Example 3: The introduction of gene knockout (ΔccpN) by traceless genetic manipulation see Figure 4

[0069] (1) Using the plasmid pSS as a template, primers ccpN-Del-P3 and ccpN-Del-P4 as amplification primers, PCR to obtain positive and negative screening boxes; using the B. subtilis168 genome as a template, using ccpN-Del-P1 and ccpN -Del-P2 was used as a primer, and the upstream homology arm F-2 (1110bp) containing DR sequence was obtained by PCR; B. subtilis168 genome was used as template, and ccpN-Del-P5 and ccpN-Del-P6 containing DR sequence were used as Primers, PCR to obtain the downstream homology arm B-2 (1140bp) containing the DR sequence; the upstream homology arm F-2 containing the DR sequence, the positive and negative screening box and the downstream homology arm B- 2. Using ccpN-Del-P7 and ccpN-Del-P8 as primers, obtain the target dsDNA fragment 1-B by fusion PCR;

[0070] (2) Inoculate BUK in 5mL LB liquid medium, culture overnight at 37°C and 220rp...

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Abstract

The invention discloses a traceless modification method of bacillus subtilis genome. The method disclosed by the invention is used for carrying out traceless modification on the bacillus subtilis genome by utilizing an upp positive-negative selection system. The method disclosed by the invention utilizes a ComK expression system, so that the bacillus subtilis has an excellent dsDNA (double-stranded deoxyribonucleic acid) conversion efficiency which can be up to 3-5*10<3>cfu/microgram dsDNA. Meanwhile, an exogenous endonuclease I-SceI expression system is utilized to bring double-stranded DNA breakage into the genome, so that the genetic recombination efficiency in negative selection molecules is obviously improved and can be up to 8*10<-4>. According to the method disclosed by the invention, iterative modification can be performed on a plurality of target nucleotide sequences in the bacillus subtilis genome, and the steps of introducing gene mutation to the genome, knocking out target gene sequences from the genome, deleting a large fragment of genomic sequences and the like are included.

Description

technical field [0001] The invention belongs to the technical field of genome transformation, and in particular relates to a method for seamlessly modifying the genome of Bacillus subtilis. Background technique [0002] Bacillus subtilis is a dominant microbial species that exists in soil and plant micro-ecosystems, and widely exists in nature. It is non-pathogenic, harmless to humans and animals, does not pollute the environment, and has strong stress resistance and antibacterial and disease-preventing effects. It is widely used as a model organism for molecular biology research and as a host for high-yield strains in industrial production. In recent years, with the rapid development of second-generation and third-generation high-throughput sequencing technologies, molecular biology research in the era of functional genomics, such as comparative genome sequencing, reverse metabolic engineering, and adaptive evolution, urgently needs a fast, seamless trace, an efficient all...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21C12R1/125
Inventor 王智文王光路石婷陈涛赵学明
Owner TIANJIN UNIV
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