Corynebacterium gene continuous knockout system, as well as construction method and application thereof

A coryneform bacterium and a construction method technology, applied in the field of microbial genetic engineering, can solve problems such as the trouble of the second round of exchange screening of sacB gene

Inactive Publication Date: 2013-11-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the insertional inactivation of the sacB gene someti...

Method used

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  • Corynebacterium gene continuous knockout system, as well as construction method and application thereof
  • Corynebacterium gene continuous knockout system, as well as construction method and application thereof
  • Corynebacterium gene continuous knockout system, as well as construction method and application thereof

Examples

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Embodiment 1

[0033] Embodiment 1 coryneform bacterium gene continuous knockout system

[0034] The coryneform bacteria gene continuous knockout system provided by the present invention includes a template plasmid and a temperature-sensitive expression plasmid; the template plasmid provides a kan fragment with loxp or mutant loxL / R sites at both ends, also known as a kan box; The sensitive expression plasmid carries the Cm resistance gene cat and the thermosensitive C. glutamicum replicon, and expresses Cre recombinase to remove the Km resistance gene kan. The template plasmid is pDTW201 / 202; the temperature-sensitive expression plasmid is pDTW109; its nucleotide sequence is shown in SEQ ID NO.1-NO.3.

[0035] The construction scheme of the coryneform bacteria gene continuous knockout system is as follows:

[0036] Step 1: Using pK18mobsacB (Schafer et al., 1994) as a template and the kan-F / kan-R primer pair as a template, PCR amplifies the kan gene, and introduces XhoI and XbaI restrictio...

Embodiment 2

[0053] Knockout of aceE in embodiment 2ATCC13032 / 14067 / 13869

[0054] 1. Obtaining the knockout plasmid pDTW301

[0055] Using the C. glutamicum ATCC14067 genome as a template, the upstream and downstream fragments of the aceE gene were amplified with corresponding primers. PstI and BamHI were introduced at the 5' and 3' ends of the upstream fragment, respectively, and XbaI and SalI restriction sites were introduced at the 5' and 3' ends of the downstream fragment, respectively. Using pDTW201 as a template, the kan box containing loxp sites at both ends was amplified with corresponding primers, and BamHI and XbaI restriction sites were introduced at the 5' and 3' ends, respectively. The upstream fragment of aceE was digested and purified with PstI and BamHI, the downstream fragment was digested and purified with XbaI and SalI, the kan box fragment was digested and purified with BamHI and XbaI, pBluescript II SK(+) was digested and purified with PstI and SalI, four fragments ...

Embodiment 3

[0071] Example 3 Knockout of ilvA in ATCC14067ΔaceE

[0072] 1. Obtaining the knockout plasmid pDTW302

[0073] Using the C. glutamicum ATCC14067 genome as a template, the upstream and downstream fragments of the ilvA gene were amplified with corresponding primers, respectively. XhoI and XbaI were introduced at the 5' and 3' ends of the upstream fragment, respectively, and PstI and BamHI restriction sites were introduced at the 5' and 3' ends of the downstream fragment, respectively. Using pDTW202 as a template, the kan box containing loxL / R sites at both ends was amplified with corresponding primers, and BamHI and XbaI restriction sites were introduced at the 5' and 3' ends, respectively. The upstream fragment of ilvA was purified by digestion with XhoI and XbaI, the downstream fragment was purified by digestion with PstI and BamHI, the kan box fragment was purified by digestion with BamHI and XbaI, pBluescript II SK(+) was purified by digestion with PstI and XhoI, four frag...

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Abstract

The invention discloses a construction method and an application of a corynebacterium gene continuous knockout system, belonging to the technical field of genetic engineering. The corynebacterium gene continuous knockout system comprises a template plasmid and a temperature-sensitive expression plasmid, wherein the template plasmid provides a kan fragment (also known as a kan box) with loxp or variant loxL/R sites at two ends; and the temperature-sensitive expression plasmid carries a Cm resistance gene cat and a temperature-sensitive C. glutamicum replicon, expresses a Cre recombinant enzyme and is used for removing a Km resistance gene kan. The corynebacterium continuous knockout system disclosed by the invention can be generally used for continuous gene transformation of corynebacterium and has the advantages of simplicity and convenience in operation and high efficiency. By utilizing the knockout system, gene (continuous) knockout of three major typical subspecies genomes of the corynebacterium can be successfully completed, and the system is proved to be applicable to research and production of corynebacterium metabolites.

Description

technical field [0001] The invention relates to a coryneform bacterium gene continuous knockout system and its construction method and application, in particular to a knockout system based on the combination of homologous recombination and specific site recombination, which belongs to the field of microbial genetic engineering. Background technique [0002] Corynebacteria are a class of Gram-positive bacteria belonging to the actinomycetes with moderate to high GC content. Since Corynebacterium glutamicum was first isolated to produce L-glutamate (Kinoshita et al., 1985), three major representatives of Corynebacterium: Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum, have been It is widely used in the production of amino acids. [0003] Since a large amount of knowledge on the physiology, biochemistry and genetics of C. glutamicum has accumulated (Eggeling and Bott, 2005; Burkovski, 2008), metabolic engineering has replaced classical ran...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12N15/09C12N1/20C12R1/13C12R1/15
Inventor 王小元胡瑾瑜李颜颜谭延振李烨
Owner JIANGNAN UNIV
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