Plate Lysates Research Articles

Aqueous two-phase systems for the recovery and purification of phage therapy products: Recovery of salmonella bacteriophage ϕSan23 as a case study

Contamination with Salmonella can cause significant economic and public health problems. Its prevention is accomplished by the use of antibiotics, but appearance of resistant strains has raised the need to search for alternatives. Recently, Salmonella-specific bacteriophages were identified and suggested as a therapy product in poultry. The large-scale production of this product requires the development of recovery/purification strategies to maximize product quality and minimize production costs. Aqueous two-phase systems (ATPS) have been used for different bioproducts, but few reports exist for the recovery of phages with this technique. This work analyzed the use of PEG-Salt and Ionic Liquid-Salt systems to characterize the partition of three phages samples: (1) Phage crude lysate containing phages, cell debris and bacterial cells (2) Centrifuged lysate containing phages, cell debris but reduced bacteria due to a centrifugation process (3) Filtered Lysate Phage containing phages and cell debris, without bacterial cells. Phage infectivity was measured in each of the ATPS components to determine the optimal system through recovery, purification fold and stability. Results indicate that phages partition to PEG 400 or ionic liquid phases, but in a PEG8000-Salt system, partition is favored to the salt-rich phase (55 and 3000-fold for phage crude lysate and filtered plate lysate, respectively) with a high recovery (60 and 90% for the same samples). Moreover, this salt phase provided the largest purification fold (up to 127-fold) and no significant reduction in infectivity. These findings serve as a platform for potential scale-up of bioprocesses for the recovery/purification of phage therapy products.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Rapid Analysis of Bacteriophage λ Isolates: Purification of λ DNA from Plate Lysates

Rapid Analysis of Bacteriophage λ Isolates: Purification of λ DNA from Plate Lysates

Adhesion of neutrophils to cultured human endothelial cells is enhanced by stimulation of adenosine A1-receptors

Adenosine exerts a variety of partly divergent effects during postischemic reperfusion. Since the intravascular retention of neutrophils (PMN) is a crucial step in the development of organ reperfusion injury, we tested whether stimulation of human endothelial adenosine A 1 -receptors enhances adhesion of PMN, as described previously for guinea pig coronary and feline coronary and pulmonary endothelium. Cultured human umbilical vein endothelial cells (HUVECs) were stimulated with different concentrations (0.01-10 μM) of the A 1 -receptor agonist N6-cyclopentyladenosine (CPA), thrombin (1 U/ml) serving as control stimulus. Adhesion of isolated human PMN on HUVECs was studied by photometrically determining the amount of the phagocyte-specific enzyme myeloperoxidase in culture plate lysates after sedimentation of PMN and removal of nonadherent PMN. The expression of CD11b, a PMN adhesion molecule and activation marker, was measured flow cytometrically on nonadherent PMN. The presence of the endothelial adhesion molecule P-selectin was also quantified by flow cytometry. Adhesion rose concentration-dependently after stimulation with CPA, reaching a maximum (55% increase vs. control) at 1 μM, then declining to 18% below control at 10 μM. Thrombin increased adhesion by 69%. CD11b on neutrophils rose after contact with the supernatant of stimulated endothelial cells, with a maximum increase of 136% at 1 μM CPA (thrombin: + 54%). P-selectin was upregulated on HUVECs upon stimulation: 25, 46, 159, and 29%, respectively, for 0.01, 0.1, 1, and 10 μM CPA (thrombin: 73%). Thus, activation of human endothelial adenosine A 1 -receptors, e.g., by CPA, causes increased adhesion and activation of PMN, and enhances expression of endothelial P-selectin. CPA partially reverses its proinflammatory actions due to a loss of specificity at high concentrations (10 μM).

A Method for Quantitative Recovery of DNA from Plate Lysates of Bacteriophage λ-Derived Clones

A method is described for the purification of DNA from recombinant clones in λ vectors. Plate lysates are used as the starting material, with the phage suspension being either loaded directly onto mini-cesium chloride two-step gradients or first subjected to precipitation with polyethylene glycol-8000 before application to the gradients. A single centrifugation step is used and the DNA is isolated from the harvested bacteriophage by incubation at room temperature with formamide followed by ethanol precipitation. The small-volume gradients were effective in isolating phage particles in the range of 1.0 to 12.5 × 1011 pfu with quantitative recovery of approximately 7 μg DNA/1011 pfu. The method was tested with a cDNA clone in λgt10 (as an example of a λ-derived vector which gives a plate lysate with a high titer) and rat genomic fragments cloned into EMBL3A (for which the plate lysates are of low titer). For both vectors, the purified DNA appeared free of inhibitory contaminants as assessed by restriction endonuclease digestion and subcloning of fragments.

Quick and efficient method for recovering of bacteriophages from soft agar after their propagation by the plate lysate technique.

Quick and efficient method for recovering of bacteriophages from soft agar after their propagation by the plate lysate technique.

Direct sequencing of lambda DNA from crude lysates using an improved linear amplification technique

We described an improved method for directly sequencing lambda (λ) DNA that has been isolated from either crude cleared lysates or plate lysates. This protocol does not require that the DNA be obtained from bacteriophage particles that have been purified by caesium chloride centrifugation. Nanogram quantities of λ DNA are unidirectionally amplified using a radioactively-labelled oligonucleotide primer, and Thermus aquaticus (Taq) DNA polymerase, in the presence of T4 gene 32 protein (gp 32). The amplification/sequencing reactions are then incubated with terminal deoxynucleotidyl transferase (TdT) and all four deoxynucleotide triphosphates to elongate any prematurely-arrested products. This procedure, which is a modification of a previously-published method, results in a significant improvement in the quality and amount of DNA sequence information that can be obtained from λ templates. Although it was developed to sequence DNA directly from λEMBL3 recombinants, it can also be used with cosmid DNA, M13 and plasmid DNA, and polymerase chain reaction (PCR) amplification products, yielding excellent ladders in each case. In addition, our method resolves the nucleotide sequences of double-stranded plasmid templates that are difficult to determine by conventional dideoxynucleotide sequencing protocols because of 'stalling', in which bands appear at the same position in all four lanes.

Preparation of Templates for DNA Sequencing

This unit contains protocols for preparing DNA suitable for use as dideoxy sequencing templates and as material for end labeling and chemical sequencing. In all protocols, the starting material contains the recombinant molecule to be sequenced. DNA from M13mp-derived phage is easily prepared and is currently the most reliable source of template for large-scale dideoxy sequencing projects. Because it is occasionally necessary or convenient to use a lambda-derived phage as a source of DNA, a protocol for preparing lambda phage DNA from plate lysates is provided. Two protocols for minipreps of plasmid DNA are provided, one intended for dideoxy sequencing, the other for end labeling and chemical sequencing; they differ primarily in the way in which cellular RNA is removed. Alkali denaturation of double-stranded DNA (necessary prior to annealing) is described, and a final protocol describes the preparation of template for thermal cycle sequencing from a single phage plaque or bacterial colony.

Filter-supported preparation of λ phage DNA

A rapid and simple method is described for the isolation of DNA from phage λ which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram qquantities of λ DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. λ phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 m sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.

A new and fast method for preparing high quality lambda DNA suitable for sequencing.

A method is described for the rapid purification of high quality lambda DNA. The method can be used from either liquid or plate lysates and on a small scale or a large scale. It relies on the preadsobtion of all polyanions present in the lysate to an "insoluble" anion-exchange matrix (DEAE or TEAE). Phage particles are then disrupted by combined treatment with EDTA/proteinase K and the resulting DNA is precipitated by the addition of the cationic detergent cetyl (or hexadecyl)-trimethyl ammonium bromide-CTAB ("soluble" anion-exchange matrix). The precipitated CTAB-DNA complex is then exchanged to Na-DNA and ethanol precipitated. The resultant purified DNA is suitable for enzymatic reactions and provides a high quality template for dideoxy-sequence analysis.

A rapid method for preparing phage λ DNA from agar plate lysates

We have developed a simple and rapid method to prepare phage λ DNA from agar-plate lysates. This method does not require purification of phage particles. Fifteen milliliters of lysate obtained from agar plates yields about 5 μg DNA. The enzyme inhibitors in this preparation are removed by an Elutip-d column. The DNA can be cut by various restriction enzymes and ligated.