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P1-35 expression units

A technology of activity and promoter, applied in the field of P1-35 expression unit, can solve the problem of not showing enhanced gene expression

Inactive Publication Date: 2007-06-20
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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  • P1-35 expression units
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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0466] Embodiment 1: Construction of vector pSK1Cat

[0467] 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 religated. The resulting plasmid was named pMT1-del. The vector pMT1-del was digested with restriction enzymes BglII and XbaI. The 2.5 kb fragment contained the pSR1 ori from C. glutamicum and was ligated to the 2 kb plasmosonpTnMod-Okm (Dennis and Zylstra (1998) Appl. Environ. Microbiol. 64:2710-2715), which had also been cut with BglII and Xbal. The resulting vector was named pSK1. The fragment of plasmposon pTnMod-Okm has the pMB1 origin of replication from E. coli and a kanamycin resistance marker (Tn903). By polymerase chain reaction (PCR) according to the standard method as described in Innis et al. (1990) PCR Protokols, A Guide to Methods and Applications, Academic Press, by oligonucleotide primer A (SEQ.ID.NO.4 ) and B (SEQ.ID.NO.5), using th...

Embodiment 2

[0472] Example 2 Plasmid pSK1P tac build

[0473] Plasmid pKK223-3 SEQ.ID.NO.6 contains (P tac )Promoter. The promoter was isolated by digestion with the restriction enzyme BamHI, and the fragment was cloned into the BamHI-linearized vector pSK1Cat SEQ ID. The plasmid was named pSK1P tac (figure 2).

Embodiment 3

[0474] Example 3P 1-35 Cloning of (SEQ.ID.NO.1)

[0475] Chromosomal DNA of C. glutamicum AS019E12 was isolated from late exponential phase cells using the method of Eikmanns et al. (1994) Microbiology 140: 1817-1828 and subsequently partially digested with the restriction enzyme Sau3AI. The resulting fragment (0.4-1.0 kb in size) 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 from individual colonies grown on these plates were isolated and analyzed. One such plasmid is pSK1Cat P 1-35 , which contains the promoter P 1-35 (SEQ. ID. NO. 1). This promoter is located upstream of the gene encoding the ABC transporter. The insert size is 280bp.

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Abstract

The invention relates to the use of nucleic acid sequences for regulating the transcription and expression of genes, to the novel promoters and expression units as such, to methods for modifying or occasioning the transcription rate and / or expression rate in genes, to expression cassettes containing said expression units, to genetically modified microorganisms having a modified or occasioned transcription rate and / or expression rate, and to methods for producing biosynthesis products by cultivating the genetically modified microorganisms.

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 . These substances are collectively referred to as fine chemicals / proteins, which include, inter alia, organic acids, proteinogenic and non-proteino...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N5/10C12N15/77C07K14/34C12P13/08C12P13/12C12P13/04
CPCC07K14/34C12N5/10C12P13/04C12N15/77
Inventor J-S·乔W·K·曾I·K·基姆S·H·利姆H-S·李
Owner BASF AG