Psod expression unit
A nucleic acid, active technology, applied in the fields of biochemical equipment and methods, applications, and botanical equipment and methods, which can solve problems such as enhanced expression of undisplayed genes
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Embodiment 1
[0476] With the aid of heterologous expression unit Pgro (SEQ.ID.2), an integrated plasmid for overexpression of pycA gene was prepared
[0477] In order to amplify the promoter of the gene encoding the chaperonin Gro ES, the following oligonucleotides are defined.
[0478] SEQ.ID.NO 5:
[0479] gro3: 5’-gccgcagcaaacccagtag-3’
[0480] SEQ.ID.NO.6:
[0481] gro11: 5’-agtcgacacgatgaatccctccatgagaaaa-3’
[0482] This primer was used in PCR reactions with chromosomal DNA from Corynebacterium glutamicum ATCC 13032. In this way, it is possible to amplify a DNA fragment equivalent to the expected size (427 bp).
[0483] In order to amplify a part of the gene encoding pyruvate carboxylase, the following oligonucleotides are defined.
[0484] SEQ.ID.NO.7:
[0485] pyc6: 5’-tttttctcatggagggattcatcgtgtcgactcacacatcttcaacgcttccag-3’
[0486] SEQ.ID.NO.8:
[0487] pyc3: 5’-cccgcagcaacgcacgcaagaaa-3’
[0488] This primer was used in PCR reaction together with chromosomal DNA from Corynebacteri...
Embodiment 2
[0507] Preparation of vector pCLiK5MCS
[0508] First, the oligonucleotide primers SEQ ID NO: 15 and SEQ ID NO: 16 were used to amplify the ampicillin resistance and the replication origin of the vector pBR322 by polymerase chain reaction (PCR).
[0509] SEQ ID NO: 15
[0510] 5’-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3’
[0511] SEQ ID NO: 16
[0512] 5’-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTCG-3’
[0513] In addition to the complementary sequence of pBR322, the oligonucleotide primer SEQ ID NO: 15 contains restriction endonucleases SmaI, BamHI, NheI and AscI along the 5'-3' direction. The oligonucleotide primer SEQ ID NO: 16 contains restriction endonucleases XbaI, XhoI, NotI and DraI along the 5'-3' direction. The PCR reaction is carried out by the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)) with PfuTurbo polymerase (Stratagene, La Jolla, USA), for example. Use GFX according to the produc...
Embodiment 3
[0541] Preparation of plasmid PmetA metA
[0542] Chromosomal DNA was prepared from Corynebacterium 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: 24 and SEQ ID NO: 25, chromosomal DNA as a template, and Pfu Turbo polymerase (available from Stratagene), as described by Innis et al. (1990) PCR Protocols. A Guide to Methods and Applications, Academic Press describes the standard method to amplify the metA gene including the 5'non-coding region by polymerase chain reaction (PCR).
[0543] SEQ ID NO: 24
[0544]5’-GCGCGGTACCTAGACTCACCCCAGTGCT-3’
[0545] and
[0546] SEQ ID NO: 25
[0547] 5’-CTCTACTAGTTTAGATGTAGAACTCGATGT-3’
[0548] Use GFX according to the product manual TM PCR, DNA and gel band purification kit (Amersham Pharmacia, Freiburg) purified DNA fragments of about 1.3 kb in size. Then cut the DNA fragment with restriction enzymes Asp718 and SpeI (Ro...
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