Production of Pcb01, a Plasmid for Dna Immunization Against the Adhesin of Escherichia Coli K88ab

Abstract

Growth characteristics and plasmid yields of Escherichia coli JM109 transformed with pCB01, a plasmid that encodes genes for the fimbrial adhesin of E. coli K88ab and for ampicillin resistance, grown in two culture media in agitated flasks and in fermentor, are reported. The rate of plasmid loss during growth was estimated by the differential counts in media with and without ampicillin. Plasmid yields of cultures grown in flasks varied from 0.9 to 67 µg/ml of medium, while those grown in fermentor attained 62 µg/ml of medium after 8 hours of culture. Plasmid bearing cells were outgrown by plasmid free cells in proportions varying from 5 to 1.2 non-transformed for each transformed cell during growth. Generation times of total population and plasmid bearing cells were 33 and 61 minutes, and 36 and 121 minutes, for fermentor and flask grown cultures, respectively. The same culture grown in fermentor and in flasks produced 62 and 33 µg of plasmid DNA per ml of medium, respectively. Bacterial concentrations and plasmid yields were higher in BHI than in LB medium. Yields of plasmid DNA obtained from the same batch were 1,7 times higher with cesium chlorideethidium bromide gradients than with commercial columns.

Interspecific Transfer of Bacterial Endosymbionts between Tsetse Fly Species: Infection Establishment and Effect on Host Fitness

“…This phenomenon was unlikely to have resulted from loss of the eGFPuv plasmid during the 30-day pupal stage (on the contrary, unpublished data from our laboratory indicated that recSodalis bacteria grown in vitro retained their plasmids in the absence of antibiotic selection over a 3-month test period). Oxygen deprivation in the sealed environment of the tsetse fly's pupal case more likely accounted for our inability to consistently detect this gene (although very faint bands were present in some samples), as the replication and stability of plasmids carrying ColE1 replication origins (i.e., pGFPuv) can be severely impeded under such conditions (15,20). We are currently developing a method of inserting exogenous DNA directly into the Sodalis chromosome via homologous recombination.…”

Labeling of prokaryotic mRNA in live cells using fluorescentin situhybridization of transcript-annealing molecular beacons (FISH-TAMB)

“…E. coli mcrA + grew at μ control = 0.16 h -1 , μ FISH-TAMB = 0.14 h -1 whereas E. coli pmoA + grew at μ control = 0.14 h -1 , μ FISH-TAMB = 0.12 h -1 . These decreased growth rates relative to plasmid-free E. coli (Sezonov et al , 2007; Gil-Turnes et al , 2001) indicated growth inhibitory effects of the mcr A and pmo A inserts and hinted the difficulty of expressing mcr A in E. coli for FISH-TAMB detection. Doubling times for both control and FISH-TAMB-treated M. barkeri were ∼ 21 hours (μ = 0.03 h -1 ), which are consistent with previous reports of hydrogenotrophic M. barkeri growth (Maestrojuan and Boone, 1991).…”