The Construction Of ATPase Gene Mutant Plasmid And Its Influence On The Growth Of Escherichia.Coli

Adenosine triphosphate (ATP) is a high-energy phosphate compounds in various cells. It provides direct energy for life activities. ATP is synthesized from ADP and phosphateinos by ATP synthase. ATP plays the vital role in energy storage and transportation, the synthesis of saccharides, fat, protein and nucleic acid. ATP provides direct energy for life activities. So it is known as molecular egergy currency. In this study, we construct a plasmid pBB-ATP which contains F-ATPase β subunit gene mutation (F-ATPase p subunit gene mutation will lead to F-ATPase activity to decrease) through homologous recombination by Saccharomcyes cerevisiae. Then the plasmid was transformed into Escherichia coli. We studied the growth curve and respiration rate of Escherichia coli to explore the effect of impairment of ATP synthesis on Escherichia coli, and provide tools to study the changes of its metabolic pathway.In this paper, the replicon (PBBR1) of broad host DNA fragment, KanR DNA fragment, and yeast replicon (2μ) fragment were obtained by PCR from plasmid pBBR1MCS5, pET29a and pXZ198. These fragments contain homologous 30bp at both ends for recomibnation. The three fragments were electroporated into Saccharomcyes cerevisiae. The shuttle plasmid pXZ200 was obtained through homologous recombination by Saccharomyces cerevisiae. By the designed mutational sites in primers. F-ATPase β subunit gene mutation fragment was obtained by PCR. The shuttle plasmid pXZ200 was linearized by enzyme. The PCR fragments and linearized pXZ200 were transformed into Saccharmcyes cerevisiae. The plasmid pBB-ATP was obtained through homologous recombination by Saccharomcyes cerevisiae.Last the plasmid pBB-ATP was transformed into Escherichia coli and the growth curve and respiration rate of Escherichia coli were studied.Results indicate that the plasmid can be obtained efficiently through homologous recombination by Saccharomcyes cerevisiae. The pXZ200 can stably replicate in Escherichia coli and pseudomona putida KT2440. The plasmid pBB-ATP has obvious effects on Escherichia coli. Escherichia coli with the plasmid grows more slowly, has a faster specific respiration rate than its wild counterpart. These demonstrate that plasmid pBB-ATP changed the metabolic pathway of bacteria...