Characteristics of Two Types of in vitro Constructed Plasmid Vectors for Bacterium Erwinia citreus
Maja Bilić, Jasenka Korajlija, Suzana Mamić and Vladimir Delić
PLIVA d.d., Research Institute, Zagreb, Croatia
Received October 18, 1994
Accepted February 28, 1995
Two types of plasmids were used for construction of cloning vectors for bacterium Erwinia citreus: cryptic plasmid denoted pP7G500 (3.8 kb) isolated from E. citreus ATCC 31623 and plasmid pUC19. Cryptic plasmid pPZG500 was inserted in plasmid pSELECT TM-1 which confers tetracycline resistance gene. Three new recombinant plnsmids were obtained and denoted pPZG501A, pPZG501B and pPZG5OlAA according to the orientation of pPZG5OO in pSELECT TM-1. Their orientation was proved by restriction analysis. All of the recombinant plasmids after transformation expressed tetracycline resistance in E. citreus. Vor further studies pPZG5O1AA (13.3 kb) was used which surely confers an intact replicón of native plasmid pPZG500. As an additional antibiotic resistance marker, fragment of 906 bp from plasmid pE194 harboring erythromycin resistance gene toas inserted into multicloning site of plasmid pUC19. New recombinant plasmid pPZG650 (3.5 kb) after transformation expressed erythromycin resistance in E. citreus. Structure of recombinant plasmid was proved by Southern hybridization and restriction analysis. It was shown that both cloned markers (TcR and ErR) are suitable for recombinant selection. Studies of plasmid stability of two constructed vectors have shown that pPZG501AA segregates stable during a great number of generations, whereas plasmid pPZG650 is gradually disappearing from the population.
Part of this paper was presented at the 2nd Croatian Congress of Food Technologists, Biotechnologists and Nutritionists, Zagreb, June15-17,1994