Pgro expression units
A nucleic acid and activity technology, applied in the field of PGRO expression unit, can solve the problem of enhanced expression of undisplayed genes
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Embodiment 1
[0458] Preparation of integrated plasmids for overexpression of the pycA gene with the aid of the heterologous expression unit Pgro (SEQ.ID.2)
[0459] To amplify the promoter of the gene encoding the chaperonin Gro ES, the following oligonucleotides were defined.
[0460] SEQ.ID.NO 5:
[0461] gro3: 5'-gccgcagcaaacccagtag-3'
[0462] SEQ.ID.NO.6:
[0463] gro11: 5'-agtcgacacgatgaatccctccatgagaaaa-3'
[0464] This primer was used in a PCR reaction with chromosomal DNA from C. glutamicum ATCC 13032. In this way it was possible to amplify a DNA fragment corresponding to the expected size (427 bp).
[0465] To amplify a portion of the gene encoding pyruvate carboxylase, the following oligonucleotides were defined.
[0466] SEQ.ID.NO.7:
[0467] pyc6: 5'-tttttctcatggagggattcatcgtgtcgactcacacatcttcaacgcttccag-3'
[0468] SEQ.ID.NO.8:
[0469] pyc3: 5'-cccgcagcaacgcacgcaagaaa-3'
[0470] This primer was used in a PCR reaction with chromosomal DNA from C. glutamicum ATCC1303...
Embodiment 2
[0489] Preparation of vector pCLiK5MCS
[0490] First, ampicillin resistance and the origin of replication of vector pBR322 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers SEQ ID NO: 15 and SEQ ID NO: 16.
[0491] SEQ ID NO: 15
[0492] 5'-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3'
[0493] SEQ ID NO: 16
[0494] 5'-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGGGCCTCG-3'
[0495] In addition to the complementary sequence of pBR322, oligonucleotide primer SEQ ID NO: 15 contains restriction endonuclease SmaI, BamHI, NheI and AscI restriction endonuclease sites along the 5'-3' direction, oligonucleotide primer SEQ ID NO: 15 ID NO: 16 contains restriction endonucleases XbaI, XhoI, NotI and DraI along the 5'-3' direction. PCR reactions are performed with PfuTurbo polymerase (Stratagene, La Jolla, USA) by, for example, the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, Academic Press (1990)). Us...
Embodiment 3
[0523] Preparation of plasmid PmetA metA
[0524] 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: 24 and SEQ ID NO: 25, chromosomal DNA as template and Pfu Turbo polymerase (obtained from Stratagene), by PCR Protocols. A Guide to Methods and The standard method described in Applications, Academic Press amplifies the metA gene including the 5' non-coding region by polymerase chain reaction (PCR).
[0525] SEQ ID NO: 24
[0526] 5'-GCGCGGTACCTAGACTCACCCCAGTGCT-3'
[0527] and
[0528] SEQ ID NO: 25
[0529] 5'-CTCTACTAGTTTAGATGTAGAACTCGATGT-3'
[0530] Use GFX according to product instructions TM A DNA fragment of about 1.3 kb in size was purified by PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg). Then cut the DNA fragment with restriction enzymes Asp718 and SpeI (Roche Diagnostics, Mannhe...
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