P19 expression units

A technology of nucleic acid and activity, applied in the direction of peptide, DNA preparation, and introduction of foreign genetic material using vectors, etc., can solve the problems of not showing enhanced gene expression, etc.

Inactive Publication Date: 2007-06-27
BASF AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In contrast, transformed cells grown on glucose did not show enhanced expression of this reporter gene

Method used

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  • P19 expression units
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0465] Embodiment 1: Construction of vector pSK1Cat

[0466] Shuttle vector pMT1 (Follettie et al. (1993) J. Bacteriol. 175:4096-4103) was digested with restriction enzymes XhoI and BamHI, then treated with Klenow fragment and ligated again. The resulting plasmid was named pMT1-del. Vector pMT1-del was digested with restriction enzymes BglII and XbaI. A 2.5 kb fragment containing the pSR1 origin of replication from C. glutamicum was ligated into a 2 kb plasmoson pTnMod-Okm also digested with BglII and XbaI (Dennis and Zylstra (1998) Appl. Environ. Microbiol. 64:2710 -2715). The resulting vector was named pSK1. The plasmposon pTnMod-Okm fragment carries the pMB1 origin of replication of Escherichia coli and a kanamycin resistance marker (Tn903). According to the standard method described in Innis et al. ((1990) PCR Protokols, A Guide to Methods and Applications, Academic Press), the cat gene without promoter was amplified by polymerase chain reaction, and the oligonucleotid...

Embodiment 2

[0471] Example 2. Plasmid pSK1P tac build

[0472] Plasmid pKK223-3 SEQ.ID.NO.6 contains (P tac )Promoter. The promoter was isolated by using the restriction enzyme BamHI, and the fragment was cloned into the vector pSK1Cat linearized with BamHI, and the plasmid was named pSK1P tac (figure 2).

Embodiment 3

[0473] Example 3.P 19 Cloning of (SEQ.ID.NO.1)

[0474] Chromosomal DNA of Corynebacterium glutamicum AS019E12 was isolated from cells in late exponential phase using the method of Eikmanns et al. ((1994) Microbiology 140: 1817-1828), and then partially digested with restriction enzyme Sau3AI. The resulting 0.4-1.0 kb fragment was ligated into the vector pSK1Cat which had been linearized with the restriction enzyme BamHI. The ligation mixture was transformed into C. glutamicum AS019E12 by electroporation using the method of Follettie et al. ((1993) J. Bacteriol. 175:4096-4103). Cells were plated on plates containing 5 μg / ml chloramphenicol. Plasmids of individual clones grown in these plates were isolated and analyzed. A plasmid containing the promoter P 19 pSK1CatP of (SEQ.ID.NO.1) 19 . This promoter is located in the upstream region of the gene encoding the putative membrane protein. The insert size is 229bp.

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Abstract

The invention relates to the use of nucleic acid sequences for regulating the transcription and expression of genes, the novel promoters and expression units therefor, methods for modifying or generating the transcription rate and / or expression rate of genes, expression cassettes containing the expression units, genetically modified micro-organisms with modified or generated transcription rates and / or expression rates, and methods for producing biosynthetic products by cultivating the genetically modified micro-organisms.

Description

technical field [0001] The present invention relates to the use of nucleic acid sequences for regulating gene transcription and expression, new promoters and expression units thereof, methods for changing or affecting gene transcription rate and / or expression rate, expression cassettes comprising the expression unit, having altered Genetically modified microorganisms with or affected transcription rate and / or expression rate and methods for producing biosynthetic products by culturing the genetically modified microorganisms. Background technique [0002] Various biosynthetic products such as fine chemicals, especially such as amino acids, vitamins and proteins are produced in cells through natural metabolic processes and are used in many industrial fields including food, feed, cosmetics, feed, food and pharmaceutical industries middle. These substances are collectively referred to as fine chemicals / proteins, which include, inter alia, organic acids, proteinogenic and non-pr...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/09C12N15/63C12N15/67C12N15/77C12P13/08C12P13/12
CPCC07K14/34C12P13/08C12N15/77C12P13/12C12N15/09C12N15/10C12N15/113
InventorJ-S·崔W·K·郑I·K·金S·H·林H-S·李
OwnerBASF AG