P EF-TU expression units

A nucleic acid, active technology, applied in the field of PEF-TU expression unit, can solve the problem of not showing gene expression enhancement

Inactive Publication Date: 2007-01-10
PAIK KWANG IND
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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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0454] Embodiment 1: Preparation of vector pCLiK5MCS

[0455] First, the ampicillin resistance gene and the origin of replication of the vector pBR322 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers SEQ ID NO: 5 and SEQ ID NO: 6.

[0456] SEQ ID NO: 5:

[0457] 5'-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3'

[0458] SEQ ID NO: 6:

[0459] 5'-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTCG-3'

[0460] The oligonucleotide primer SEQ ID NO: 5, in addition to the sequence complementary to pBR322, also contains restriction endonucleases SmaI, BamHI, NheI and AscI along the 5'-3' direction. The nucleotide primer SEQ ID NO: 6 not only contains the sequence complementary to pBR322, but also contains restriction endonucleases XbaI, XhoI, NotI and DraI along the 5'-3' direction. PCR reactions are performed with PfuTurbo polymerase (Stratagene, LaJolla, USA) by, for example, the standard method of Innis et al. (PCR Protocols. A Guide ...

Embodiment 2

[0487] Embodiment 2: the preparation of plasmid PmetA metA

[0488] Chromosomal DNA was prepared from C. glutamicum ATCC 13032 as described in Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828. Using oligonucleotide primers SEQ ID NO: 14 and SEQ ID NO: 15, chromosomal DNA as template and Pfu Turbo polymerase (from Stratagene), PCR Protocols. A Guide to Methods and Applications , Academic Press to amplify the metA gene including the 5' non-coding region by polymerase chain reaction (PCR).

[0489] SEQ ID NO: 14:

[0490] 5'-GCGCGGTACCTAGACTCACCCCAGTGCT-3'

[0491] SEQ ID NO: 15:

[0492] 5'-CTCTACTAGTTTAGATGTAGAACTCGATGT-3'

[0493] Use GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) were used to purify the obtained DNA fragment with a size of about 1.3 kb according to the product instructions. Then cut the DNA fragment with restriction enzymes Asp718 and SpeI (Roche Diagnostics, Mannheim), and use G...

Embodiment 3

[0498] Embodiment 3: Preparation of plasmid pCLiK5MCS P EF-TU metA

[0499] Chromosomal DNA was prepared from C. glutamicum ATCC 13032 as described by Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al. (1994) Microbiology 140: 1817-1828. Using oligonucleotide primers SEQ ID NO: 17 and SEQ ID NO: 18, chromosomal DNA as template and Pfu Turbo polymerase (from Stratagene), by, for example, Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications , a standard method of Academic Press to amplify a DNA fragment of about 200 bp of the 5' non-coding region (promoter region) (Psod) of superoxide dismutase by polymerase chain reaction (PCR).

[0500] SEQ ID NO: 17:

[0501] 5'-GAGACTCGAGGGCCGTTACCCTGCGAATG-3'

[0502] SEQ ID NO: 18:

[0503] 5'-CCTGAAGGCGCGAGGGTGGGCATTGTATGTCCTCCTGGAC-3'

[0504] Use GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg) were used to purify the obtained DNA fragments according to the product instructions. ...

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Abstract

The invention relates to the use of nucleic acid sequences for regulating gene transcription and expression, said novel promoters and expression units, methods for modifying or inducing the gene transcription rate and / or expression rate, expression cassettes containing said expression units, genetically modified microorganisms having a modified or induced transcription rate and / or expression rate, and methods for producing biosynthetic products by cultivating said 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, such as amino acids, vitamins and proteins among others, are produced in cells through natural metabolic processes and are used in many industrial fields including cosmetic, feed, food and pharmaceutical industries. These substances are collectively referred to as fine chemicals / proteins, which include, inter alia, organic acids, proteinogenic and non-proteinogenic amino ...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N15/77C07K14/34C12N1/21C12NC12N1/00C12N15/63C12N15/67C12P1/00C12P13/08C12P13/12
CPCC07K14/34C12P13/08C12N15/77C12P13/12C12N15/63C12N15/67
InventorB·克勒格尔O·策尔德尔C·克洛普罗格H·施罗德S·哈夫纳
OwnerPAIK KWANG IND