Research techniques
We use a combination of molecular biology, microbiology and microscopy in our research. Some of the main techniques we use are described below.
Investigating microbial communities in activated sludge
A huge variety of bacteria and other microbes live in activated sludge. We investigate the abundance, diversity and structure of these bacterial communities using a combination of PCR, quantitative real-time PCR, clone library analysis and DNA sequencing, and fluorescent in-situ hybridisation (FISH).
PCR (polymerase chain reaction)
PCR is a very important tool in molecular biology research, in which an enzyme (DNA polymerase) is used to copy a piece of DNA up to a billion times in a molecular chain reaction. This makes it possible to analyse the DNA through techniques such as DNA sequencing.
A PCR reaction typically includes the DNA polymerase enzyme, a buffer mixture, the components required to build the new DNA molecules, and two short DNA fragments called primers, which determine which DNA sequence is targeted for copying. The reaction mixture is subjected to a repeating temperature cycle, during which the primers bind to the target DNA and the enzyme copies the DNA sequence between the two primers.
Quantitative real-time PCR
Quantitative real-time PCR allows the amount of DNA in a sample to be accurately measured. This can be used to indicate the abundance of bacterial cells.
In quantitative real-time PCR, one of the PCR primers carries a fluorescent label. As the reaction proceeds, the fluorescently-labelled primer is incorporated into each newly copied DNA molecule, resulting in production of an increasing fluorescent signal. This signal can be measured in real-time throughout the reaction, and can be used to calculate the initial number of DNA molecules present. Depending upon the primers used, this can be used to determine the abundance of different types of bacteria in samples.
Clone library analysis and DNA sequencing
Clone library analysis and DNA sequencing are used to identify different bacteria present in environmental samples.
Bacteria have a gene called the 16S ribosomal RNA gene (or 16S rRNA gene), which can be amplified using PCR. The DNA sequence of the 16S rRNA gene varies between different species, and can therefore be used to identify bacteria, somewhat like a bar code.
PCR amplification of the 16S rRNA gene from activated sludge results in production of a mixture of different sequences from different bacteria. It is difficult to identify sequences in a mixture, so cloning is used to separate them.
To create a clone library, the mixture of 16S rRNA gene sequences is introduced to specially prepared bacteria. Individual 16S rRNA gene sequences will be taken up by bacterial cells, which can then be spread out on agar plates. Each bacterial cell will subsequently multiply and form a circular colony.
Each colony starts from a single cell, so all the bacteria in each colony should be genetically identical and contain the same cloned 16S rRNA gene sequence. PCR can then be used to amplify the cloned 16S rRNA gene from each colony, resulting in production of a series of PCR products which each contain the 16S rRNA gene sequence from a single organism, rather than the mixture of different 16S rRNA gene sequences that we started with. These PCR products can be sequenced, allowing identification of the different bacterial species that are present.
Fluorescence in-situ hybridisation (FISH)
FISH involves the use of short DNA probes, much like the primers used in PCR, which are labelled with fluorescent markers. These are designed so that they will bind to particular DNA sequences, such as those characteristic of different bacterial species.
When added to biofilm, floc or wastewater samples, the probes bind to targeted DNA sequences in the sample. Using a microscope, the location of the fluorescently-labelled primers within the sample can be observed, and indicate the location of different types of bacteria within the activated sludge community.
Sequencing the Acidovorax genome
The entire genetic material of an organism, made of DNA, is known as a genome. A genome contains the complete set of genetic information required for the development and maintenance of all the molecules, cells, tissues and processes that together constitute an organism. Obtaining the complete sequence of an organism's genome takes a lot of work, but offers the chance to access this information and gain in-depth understanding of the fundamental factors which control the nature of the organism.
We are in the final stages of sequencing the genome of Acidovorax temperans CB2, using a whole-genome shotgun sequencing approach. Further information on the steps involved in whole genome sequencing is available at http://www.jgi.doe.gov/education/how/.
Analysing gene expression in Acidovorax
When a gene is expressed, its DNA sequence is copied into messenger RNA, which acts as a template for protein synthesis. Analysing messenger RNA levels in cells and tissues therefore offers a means of determining which genes are being expressed. We investigate gene expression in different strains of Acidovorax using gene expression microarrays.
Whole genome Combimatrix gene expression microarrays
A DNA microarray is series of hundreds or thousands of different gene sequences arranged on a slide or a chip. Our microarrays, manufactured by Combimatrix, contain DNA sequences from the Acidovorax genome.
To analyse gene expression in Acidovorax, differently-coloured fluorescent labels are attached to both messenger RNA and microarray DNA sequences. The sequence of a messenger RNA molecule will bind to its corresponding DNA sequence on a microarray. The interaction of different coloured labels means that the colour detected at each microarray site varies according to the amount of mRNA bound at each site.
Comparing microarray colour profiles from different types of Acidovorax allows visualisation of differences in gene expression. We know what the DNA sequence at each spot on the microarray is, so we can identify which genes are important in the different varieties.
Analysing the proteome of Acidovorax
A proteome is the complete set of proteins—the main components of cellular machinery—expressed in a cell or tissue. Studying the proteome of Acidovorax provides the opportunity to understand its physiological and metabolic properties. We use 2D gel electrophoresis to visualise proteins, and MALDI-TOF-MS to identify them.
Proteomic analyses are being completed using facilities within the University of Auckland's Centre for Genomics & Proteomics.
2D gel electrophoresis
Electrophoresis refers to the tendency of proteins (and other molecules) to move through a gel matrix when subjected to an electrical field. Proteins vary in size and electrical charge, which affects their speed of movement. 2D gel electrophoresis is a method of separating out a mixture of proteins according to these factors.
First, a protein mixture is placed upon a polyacrylamide gel matrix containing components that produce a pH gradient when a voltage is applied. The proteins spread out in a line through this medium, until each reaches the pH where its net charge is zero.
Second, a detergent is added (SDS), which attaches to proteins at a rate directly proportional to their size. SDS causes proteins to unfold from their 3-d shapes into long, straight rods, and gives them a substantial negative charge. When a voltage is applied in a direction perpendicular to that of the first, the proteins will now migrate through the gel at a rate proportional to their size.
This results in a gel with spots of protein spread out across it in two dimensions, according to charge and size. Protein spots can be cut out for subsequent analysis.
MALDI-TOF-MS
MALDI-TOF-MS stands for Matrix-Assisted Laser Desorption Ionisation-Time of Flight-Mass Spectrometry, and allows identification of proteins. It's less complicated than it might sound.
Basically, a protein sample is mixed with a matrix that contains a light-absorbing chromophore, and crystallised on a plate. A laser is fired at the sample/matrix, which absorbs photons and transfers energy to the protein components, which are ejected into a gas phase as ions. The time-of-flight detector measures the speed at which the ions move through an electrical field, which is proportional to mass/charge ratio and can be used to identify them.
Microscope techniques
Microscopes allow us to observe bacterial cells and assemblages, and to study their structure and organisation. We use different types of microscopes at various points in our research.
Light microscopy
Light microscopes, in combination with stains and fluorescent labels, are useful for general observations of bacterial colonies and structures, but lack the resolution to allow in-depth examination of bacterial cells and structures.
Laser dissection microscopy
Bacterial cells observed in activated sludge samples can be genetically identified using laser dissection microscopy, in combination with PCR. In this process, sludge samples are placed on an inverted light microscope and the resulting images are relayed to a computer. Areas within samples can be selected for analysis using a drawing tool, and a laser is used to vapourise the selected areas. The vapourised material is collected in a small tube, and can subsequently be analysed using PCR, allowing identification of the DNA sequences present.
Confocal microscopy
Confocal laser-scanning microscopes are equipped with lasers, which are used to scan fluorescently-labelled specimens. The fluorescent signal produced is detected and assembled into an image by a computer. Any out-of-focus signal is eliminated by filters. This results in images of much improved clarity compared to those from conventional light microscopes. Confocal microscopes have the capability of obtaining successive optical sections through a sample, which can be assembled into 3-dimentional visualisations, or time-lapse image sequences. Further explanation of confocal microscopy is available from Olympus and Nikon.
Electron microscopy
Electron microscopes have much higher resolution then light microscopes, and allow observation of specially-prepared samples at magnifications of up to 100 000 times. This is sufficient to provide very high resolution images of bacterial cells and sub-cellular components. Transmission electron microscopy (TEM) is used to obtain images from very thin cross-sections, which is useful for investigating the internal structure of cells. Scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) both produce 3-dimensional images of cell surfaces and structures.