P2-10-expressionseinheiten
A nucleic acid and active technology, which is applied in the preparation of peptides and DNA, and the introduction of foreign genetic material using vectors, which can solve problems such as enhanced expression of unshown genes.
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Embodiment 1
[0469] Example 1: Construction of vector p5K1
[0470] The 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. This 2.5 kb fragment contains the pSR1 ori from C. glutamicum and is ligated to the 2 kb plasposon pTnMod-Okm (Dennis and Zylstra (1998) Appl. Environ. Microbiol. 64:2710-2715), which has also been cut with BglII and XbaI . 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). According to the standard method described in Innis et al. (1990) PCRProtokols, A Guide to Methods and Applications, Academic Press, using oligonucleotide primers A (SEQ.ID.NO.4) and B (SEQ.ID.NO.5 ), using the vector PKK232-8 (SEQ.ID.NO.3) as ...
Embodiment 2
[0474] Example 2 Plasmid pSK1P tac build
[0475] 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
[0476] Example 3P 2-10 Cloning of (SEQ.ID.NO.1)
[0477] 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 spread 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 2-10 , which contains the promoter P 2-10 (SEQ. ID. NO. 1). This promoter is located upstream of the gene encoding sugar-phosphate epimerase. The insert size is 208bp.
[0478] 4. Resistance of Corynebacterium glutamic...
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