F. Dickely et al., ISOLATION OF LACTOCOCCUS-LACTIS NONSENSE SUPPRESSORS AND CONSTRUCTIONOF A FOOD-GRADE CLONING VECTOR, Molecular microbiology, 15(5), 1995, pp. 839-847
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 s
uppressor strains and the DNA sequence determined, One suppressor is a
n ochre suppressor with an altered tRNA(gin) and the other an amber su
ppressor with an altered tRNA(ser). The nonsense suppressors allowed i
solation 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 Lactococcos and a synthetic polylinker with
11 unique restriction sites was constructed using the ochre suppressor
as a selectable marker. Selection, following electroporation of a sup
pressible purine auxotroph, can be done on purine-free medium. The pep
N gene from L. lactis Wg2 was subcloned resulting in a food-grade plas
mid giving a four- to fivefold increase in lysine aminopeptidase activ
ity.