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A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

University of Alberta

Existing models for bacteriophage adsorption are modified with the addition of a new term, adsorption efficiency, and applied to a T4-Escherichia coli system. The adsorption efficiency is the fraction of phage that adsorbs irreversibly to the host. Adsorption kinetics were modeled using the adsorption rate constant(k) and the adsorption efficiency(epsilon). Experimental data demonstrated that the adsorption rate constant depends strongly on the condition of the host while the adsorption efficiency is a property of the bacteriophage population. The adsorption efficiency exhibited a marked dependence on the concentration of L-tryptophan. The system was used to study the effect of adsorption kinetics on bacteriophage amplification. Increasing adsorption efficiency had an effect similar to increasing the initial multiplicity of infection; the number of phages produced during amplification decreased. Optimizing the adsorption efficiency by manipulating the L-tryptophan concentration yielded a 14-fold increase in the number of phages produced.

Bacteriophage adsorption efficiency and its effect on amplification.

Self-cycling fermentation (SCF), a cyclical, semi-continuous process that induces cell synchrony, was incorporated into a recombinant protein production scheme. Escherichia coli CY15050, a lac(-) mutant lysogenized with temperature-sensitive phage λ modified to over-express β-galactosidase, was used as a model system. The production scheme was divided into two de-coupled stages. The host cells were cultured under SCF operation in the first stage before being brought to a second stage where protein production was induced. In the first stage, the host strain demonstrated a stable cycling pattern immediately following the first cycle. This reproducible pattern was maintained over the course of the experiments and a significant degree of cell synchrony was obtained. By growing cells using SCF, productivity increased 50% and production time decreased by 40% compared to a batch culture under similar conditions. In addition, synchronized cultures induced from the end of a SCF cycle displayed shorter lysis times and a more complete culture-wide lysis than unsynchronized cultures. Finally, protein synthesis was influenced by the time at which the lytic phase was induced in the cell life cycle. For example, induction of a synchronized culture immediately prior to cell division resulted in the maximum protein productivity, suggesting protein production can be optimized with respect to the cell life cycle using SCF.

Self-cycling operation increases productivity of recombinant protein in Escherichia coli.

Synchronized Escherichia coli cultures were infected with bacteriophage T4 at discrete points in the cell growth cycle. The cell cycle had a significant impact on the outcome of infection. Cell burst size was smallest for newly formed cells and increased dramatically as these progressed in the cell cycle. The largest burst sizes were achieved when infecting cells immediately prior to cell division. When cells were infected during cell division, the burst size was reduced back to its initial value. Interestingly, lysis time was longest for young cells, reached a minimum at the same point that burst size reached its maximum value, and then increased at the commencement of cell division. Consequently, phage productivity in cells about to undergo cell division was almost three times greater than the productivity of young, newly formed cells. The availability of intracellular resources is believed to be the major driving force behind phage productivity during infection. Indeed, intracellular RNA contents at the time of infection were found to correlate strongly with phage productivity. There was no significant relationship between cell DNA levels and phage productivity. Finally, burst size experiments suggested that the cell cycle also influenced the likelihood of a phage to undergo productive infection.

Zachary J. Storms

Department of Chemical and Materials Engineering

University of Alberta

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Zachary J. Storms

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Chemical and Materials Engineering

University of Alberta

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Department of Chemical and Materials Engineering