Assimilable organic carbon (AOC) determination using GFP-tagged Pseudomonas fluorescens P-17 in water by flow cytometry.

One of the newly developed methods for Assimilable organic carbon (AOC) determination is leveraged on the cell enumeration by flow cytometry (FC) which could provide a rapid and automated solution for AOC measurement. However, cell samples staining with fluorescence dye is indispensable to reduce background and machine noise. This step would bring additional cost and time consuming for this method. In this study, a green fluorescence protein (GFP) tagged strain derived of AOC testing strain Pseudomonas fluorescens P-17 (GFP-P17) was generated using Tn5 transposon mutagenesis. Continuous culture of this mutant GFP-P17 showed stable expression of eGFP signal detected by flow cytometry without staining step. In addition, this GFP-P17 strain displayed faster growth rate and had a wider range of carbon substrate utilization patterns as compared with P17 wild-type. With this strain, the capability of a new FC method with no dye staining was explored in standard acetate solution, which suggests linear correlation of counts with acetate carbon concentration. Furthermore, this FC method with GFP-P17 strain is applicable in monitoring GAC/BAC efficiency and condition as similar trends of AOC level in water treatment process were measured by both FC method and conventional spread plating count method. Therefore, this fast and easily applicable GFP-P17 based FC method could serve as a tool for routine microbiological drinking water monitoring

Correction: Assimilable organic carbon (AOC) determination using GFP-tagged Pseudomonas fluorescens P-17 in water by flow cytometry.

[This corrects the article DOI: 10.1371/journal.pone.0199193.]

Representative fluorescence histogram data of GFP-P17 and P17 in water.

<p>The x-axis shows green (FITC) fluorescence intensity, and the y-axis shows the number of events recorded for a corresponding fluorescence intensity. 1, water sample without cells; 2, water sample with P17; 3, water sample with GFP-P17.</p

AOC concentration measured using flow cytometry (FC) method and spread plate counting method(SPC).

<p>AOC concentration measured using flow cytometry (FC) method and spread plate counting method(SPC).</p

Comparison of carbon substrate utilization pattern of GFP-P17 and P17.

<p>Comparison of carbon substrate utilization pattern of GFP-P17 and P17.</p

Batch growth curves of GFP-P17 and P17.

<p>Growth conditions: 100 µg/L of acetate carbon with supplement of mineral salts. Error bars indicate standard deviation on triplicate samples.</p

Schematic presentation of our method compared to other methods.

<p>A. Conventional method; B, Flow cytometric method with cell staining. C, Our flow cytometric method without cell staining.</p

Cell counts for growth of GFP-P17 cells on a defined carbon source (sodium acetate).

<p>Cell counts for growth of GFP-P17 cells on a defined carbon source (sodium acetate).</p

Dot plots for GFP-P17 cells grown on water of 100 µg/L of acetate carbon with supplement of mineral salts.

<p>Enumeration gates are drawn to distinguish cells from background. Data was recorded in one minute. Cell counts: Day 0, 457/mL; Day 2, 3647/mL; Day 3, 80737/mL; Day 4, 61508/mL.</p

A universal method for fishing target proteins from mixtures of biomolecules using isothermal titration calorimetry

The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization