Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics

Bacterial cultures - A service discontinued

It was announced recently by both press and radio that the Department of Agriculture, which for nearly 30 years, has been supplying farmers with bacterial cultures for leguminous seed inoculation, has discontinued this service. The decision was made because adequate supplies of commercial peat cultures are now available through ordinary commercial channels

EcoBot-II: An artificial agent with a natural metabolism

In this paper we report the development of the robot EcoBot-II, which exhibits a primitive form of artificial symbiosis. Microbial Fuel Cells (MFCs) were used as the onboard energy supply, which consisted of bacterial cultures from sewage sludge and employed oxygen from free air for oxidation at the cathode. EcoBot-II was able to perform sensing, information processing, communication and actuation when fed (amongst other substrates) with flies. This is the first robot in the world, to utilise unrefined substrate, oxygen from free air and exhibit four different types of behaviour

Developing a High-Throughput LacZ Reporter Gene Assay to Evaluate Activation of the xpt-pbuX Guanine Riboswitch by Analogue Ligands

Riboswitches are special structures of RNA typically found in bacteria. They respond to the presence of certain molecules, called ligands, within the bacterial cells to regulate expression of certain genes. Genes expression is the process by which information encoded in a segment of an organism’s genetic material is used to produce functional molecules utilized by that organism. Riboswitches are currently being researched as a possible new class of antibiotic targets amid the growing public health threat of antibiotic resistance. In order to compare the effects of guanine (the natural ligand of the riboswitch), guanosine (a biological molecule similar to guanine) and SK4 (a molecule synthesized by previous Bard College seniors based on the structure of guanosine) on the xpt-pbuX riboswitch, I used a reporter gene assay in a model organism: a harmless type of bacteria called Bacillus subtilis. The structure of the xpt-pbuX riboswitch allows it to interact with ligands of shape similar to guanine. For the reporter gene assay, a gene (called LacZ) that encodes the enzyme beta-galactosidase is specially inserted into the bacterial genome next to the riboswitch. The production of this enzyme is measure by adding a chemical called ONPG to the bacterial cultures. The enzyme reacts with ONPG to produce a yellow color that can be measured. The less intense the yellow color, the less active enzyme has been produced by the bacteria. Thus, expression of the LacZ gene “reports” back on the activation of the riboswitch. If incubating bacterial cultures with a given ligand results in less production of active enzyme as compared to incubation without the ligand, this is presumed to mean that the ligand has activated the riboswitch. In this project I worked to develop an alternative method for the traditional beta-galactosidase assay, minimizing the quantities of chemicals and bacterial cultures required. Instead of incubating bacterial cultures in vials with the study ligands and using cuvettes of bacterial cultures for the enzyme assay, I incubated very small volumes of bacterial cultures in the wells of 96-well microplates and then performed the assay in 96-well microplates. The intensity of yellow color in these bacterial cultures can be directly measured in the microplates using a plate-reader. I also implemented a kinetic assay in which the changing intensity of yellow color is monitored over time instead of waiting for yellow color to develop and then measuring it, with the hope of minimizing timing error in the assay

Stable Bacterial Cultures for Producing Alginates

Methods for mass producing bacterial alginate, bacterial cultures for producing alginate, and pharmaceutical compositions containing bacterial alginate are contemplated

Paper-based sensors for rapid detection of virulence factor produced by Pseudomonas aeruginosa

Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in vitro adaptation of P. aeruginosa infections via rapid screenings of bacterial cultures that otherwise are expensive and time-consuming

Microbial diversity in heavy-metal polluted waters

Indigenious water microflora as well as the presence of metal- and xenobiotic biotransforming bacteria were investigated in waters near the KCM Pb-Zn smelter, South Bulgaria. Content of As, Hg, Cd, Mn, Pb, Cu and Zn exceeded in times the maximum permission standart. Absence of some microbial groups demonstrated a change in the microbial community structure in the region. Ecotoxicology test ISO/DIS 10712.2 displayed toxic environmental effect of the polluted waters, especially one of them which demonstrated 72 % of ecotoxicity. More than 20 ecologically relevant new bacteria were cultured. Three of them demonstrated tolerance to Cd, Cu and Mn and five- a tolerance to 2,4-dichlorphenoxyacetic acid. Our result revealed that the heavy metal pollutions reduced the microbial diversity in the studied waters, are ecotoxic as well as that some of newly isolated bacteria possess a capacity for a clean-up biotechnologies in the region. . 1, . 2, 2., 3, 3, .

Regulated bioluminescence as a tool for bioremediation process monitoring and control of bacterial cultures

An effective on-line monitoring technique for toxic waste bioremediation using bioluminescent microorganisms has shown great potential for the description and optimization of biological processes. The lux genes of the bacterium Vibrio fischeri are used by this species to produce visible light. The lux genes can be genetically fused to the control region of a catabolic gene, with the result that bioluminescence is produced whenever the catabolic gene is induced. Thus the detection of light from a sample indicates that genetic expression from a specific gene is occurring. This technique was used to monitor biodegradation of specific contaminants from waste sites. For these studies, fusions between the lux genes and the operons for naphthalene and toluene/xylene degradation were constructed. Strains carrying one of these fusions respond sensitively and specifically to target substrates. Bioluminescence from these cultures can be rapidly measured in a nondestructive and noninvasive manner. The potential for this technique in this and other biological systems is discussed