Browsing by Author "Peretti, Steven W."

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Effects of biofilm formation on plasmid segregational stability and expression
(1992) Peretti, Steven W.; Bryers, James D.
Recombinant plasmids are a significant tool for biotechnological research and have enormous commercial potential for the expression of foreign genes in procaryotic and eucaryotic organisms. Two major impcdiments to widespread utilization of genetically-engineered expression systems within natural ecosystems are: (1) the instability of the plasmid under a variety of conditions, and (2) the lack of knowledge regarding the fate of recombinant DNA in natural ecosystems. Governmental regulations have thus far severely restricted the development of processes involving the exposure to the environment of geneticallymanipulated organisms. Not surprisingly, significant effort has been spent in determining the mechanisms involved in plasmid loss from cells, and in initial studies of the fate of geneticallyengineered DNA sequences in the environment.
Effects of inducer levels on a recombinant bacterial biofilm formation and gene expression
(1994-09) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
A segregationally stable host-plasmid system, E. coli DH5α (pTKW106), was used to study the effect of induction on the accumulation rate of cells and gene expression in biofilm cultures. Isopropyl β-D-thiogalactoside (IPTG) was used to induce the expression of β-galactosidase from the plasmid. The biofilm cell net accumulation rates decreased with increasing induction levels. At 0.17 and 0.34 mM of IPTG, the biofilm cell net accumulation rates ranged between 17 and 30% when compared to the uninduced case. At 0.51 mM of IPTG, the biofilm cell density never increased. At 0.17 and 0.34 mM of IPTG, β-galactosidase contents reached maxima 36 hours after induction with both amounts representing about 7.5% of total protein. At 0.51 mM of IPTG, β-galactosidase production reached its maximum, about 16% of total protein, 48 hours after induction. The β-galactosidase mRNA synthesis rates increased with increasing inducer levels. Maximum β-galactosidase mRNA synthesis rates were reached 36 hours after induction for each IPTG concentration.
Effects of medium carbon-to-nitrogen ratio on biofilm formation and plasmid stability
(1994-07) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
Biofilm formation and plasmid segregational instability in biofilm cultures of Escherichia coli DH5α (pMJR1750) were investigated under different medium-carbon-to-nitrogen (C/N) ratios. At C/N ratios of 0.07 and 1, net accumulation of both biofilm plasmid-bearing and plasmid-free cells continued through the entire experiment without attaining any apparent steady state. At C/N ratios of 5 and 10, net biofilm cell accumulation for the two populations reached apparent steady states after 84 and 72 h, respectively. At C/N ratios of 0.07 and 1, polysaccharide production increased slowly and reached about 2g alginate equivalent/cm2 by the end of both experiments. At a C/N ratio of 5, polysaccharide increase significantly after 84 h, reaching about 7μg alginate equivalent/cm2 prior to termination. At a C/N ratio of 10, polysaccharide increased significantly after 72 h and reached 21 μg alginate equivalent/cm2 at 108 h. At C/N ratios of 0.07 and 1, protein production reached 6.5 and 4 μg/cm2, respectively. At C/N ratios of 5 and 10, protein production increased slightly for the first 84 h and reached a maximum at 108 h, at 3 and 2 μg/cm2, respectively, then decreased over the last 12 h of the experiment. Ratios of polysaccharide to protein increased with increasing C/N ratios. At C/N ratios of 0.07 and 1, the ratios between extracellular polysaccharide (EP) and protein were no more than 205 μg polysaccharide/μg protein, whereas those at C/N ratios of 5 and 10 increased to about 7 and 12 μg polysaccharide/μg protein, respectively. Probabilities of plasmid loss in the biofilm cultures increased with increasing C/N ratios. At C/N ratios of 0.07, 1, and 5, the probabilities of plasmid loss were 0.0013 ± 0.011, 0.020 ± 0.006 and 0.122 ± 0.021, respectively. At a C/N ratio of 10, the probability of plasmid loss was significantly higher, reaching 0.38 ± 0.125. The increase of probability of plasmid loss at higher C/N ratios results from competition between cell replication and extracellular polysaccharide production. © 1994 John Wiley & Sons, Inc.
Mobilization of a broad host range plasmid from pseudomonas putida to an established biofilm of bacillus azotoformans part i: experiments
(1998-02) Beaudoin, D. L.; Bryers, James D.; Cunningham, Alfred B.; Peretti, Steven W.
A strain of Pseudomonas putida harboring plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor under three different concentrations of the limiting nutrient, succinate. Experimental results demonstrated that the broad host range RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. At the lower concentrations, donor mediated plasmid transfer increased with increasing nutrient levels, but the highest nutrient concentration yielded the lowest rate of donor to recipient plasmid transfer. For transconjugant initiated transfer, the rate of transfer increased with increasing nutrient concentrations for all cases. At the lower nutrient concentrations, the frequency of plasmid transfer was higher between donors and recipients than between transconjugants and recipients. The reverse was true at the highest succinate concentration. The rates and frequencies of plasmid transfer by mobilization were compared to gene exchange by retrotransfer. The initial rate of retrotransfer was slower than mobilization, but then increased dramatically. Retrotransfer produced a plasmid transfer frequency more than an order of magnitude higher than simple mobilization.
Mobilization of a broad host range plasmid from pseudomonas putida to an established biofilm of bacillus azotoformans part ii: modeling
(1998-02) Beaudoin, D. L.; Bryers, James D.; Cunningham, Alfred B.; Peretti, Steven W.
A strain of Pseudomonas putida that harbors plasmids RK2 and pDLB101 was exposed to a pure culture biofilm of Bacillus azotoformans grown in a rotating annular reactor. Transfer of the RK2 mobilizable pDLB101 plasmid to B. azotoformans was monitored over a 4-day period. Experimental results demonstrated that the broad host range, RSF1010 derivative pDLB101 was transferred to and expressed by B. azotoformans. In the companion article to this work, the rate of plasmid transfer was quantified as a function of the limiting nutrient, succinate, and as a function of the mechanism of transfer. A biofilm process simulation program (AQUASIM) was modified to analyze resultant experimental data. Although the AQUASIM package was not designed to simulate or predict genetic events in biofilms, modification of the rate process dynamics allowed successful modeling of plasmid transfer. For the narrow range of substrate concentrations used in these experiments, nutrient level had only a slight effect on the rate and extent of plasmid transfer in biofilms. However, further simulations using AQUASIM revealed that under nutrient poor conditions, the number of transconjugants appearing in the biofilm was limited.
Plasmid retention and cloned gene expression in suspended versus biofilm cultures of e. coli dh5(pmjr1750)
(1993-01) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5α(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5α(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h−1 to 0.35 h−1 and the β-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h−1, about 36% of that without IPTG, and the β-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction. In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5β(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The β-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively.
Use of flow cell reactors to quantify biofilm formation kinetics
(1992-05) Huang, Ching-Tsan; Peretti, Steven W.; Bryers, James D.
A parallel plate flow cell reactor is introduced and used to evaluate cell adhesion and biofilm formation kinetics for four different bacterial strains of the species,E. coli. The reactor allows biofilm growth under defined, well-controlled fluid dynamics while providing continuous observations and direct sampling of biofilm for biological, chemical and physical analyses as well as immunofluorescent labeling.