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Isolation of Lactococcus lactis nonsense suppressors and construction of a food‐grade cloning vector
Author(s) -
Dickely Fraçoise,
Nilsson Dan,
Hansen Egon Bech,
Johansen Eric
Publication year - 1995
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1995.tb02354.x
Subject(s) - biology , lactococcus lactis , selectable marker , nonsense mutation , multiple cloning site , microbiology and biotechnology , plasmid , cloning vector , genetics , mutant , gene , mutation , recombinant dna , vector (molecular biology) , bacteria , lactic acid , missense mutation
Sumnnary Nonsense suppressor strains of Lactococcus lactis were isolated using plasmids containing nonsense mutations or as revertants of a nonsense auxotrophic mutant. The nonsense suppressor gene was cloned from two suppressor strains and the DNA sequence determined. One suppressor is an ochre suppressor with an altered tRNA gin and the other an amber suppressor with an altered tRNA ser . The nonsense suppressors allowed isolation of nonsense mutants of a lytic bacteriophage and suppressible auxotrophic mutants of L. lactis MG1363. A food‐grade cloning vector based totally on DNA from Lactococcus and a synthetic polylinker with 11 unique restriction sites was constructed using the ochre suppressor as a selectable marker. Selection, following etectroporation of a suppressible purine auxotroph, can be done on purine‐free medium. The pepN gene from L. lactis Wg2 was subcloned resulting in a food‐grade plasmid giving a four‐ to fivefold increase in lysine aminopeptidase activity.