Polymerase chain reaction

Event Date: 
Wednesday, January 28, 2015 - 19:00 - 19:45
Institution: 
University of Sydney
Title: 

The use of genomics in diagnostic and public health microbiology

Abstract: 

Since 2004 technological advances have enabled us to sequence more nucleic acid and generate more data in a shorter amount of time. Decreases in cost per nucleotide sequenced, the initial price of sequencing machines and the complexity of library construction means that whole genome sequencing (WGS) is available in many research labs and an increasing number of public health microbiology labs. I will examine the use of WGS in public health microbiology, particularly the possibility of investigating organisms without culture, the interrogation of genomes where PCR may be unavailable, outbreak investigation, tracking resistance mutations and novel pathogen discovery.

Event Date: 
Wednesday, July 30, 2014 - 18:15 - 18:30
Institution: 
Macquarie University
Title: 

Effect of Low Temperature on Tropical and Temperate Isolates of Marine Synechococcus.

Abstract: 

An abundant and globally occurring marine picocyanobacterium, the genus Synechococcus is an important player in oceanic primary production and global carbon cycling. In the complex marine environment, this widespread organism has evolved to successfully colonize and inhabit different environmental niches. Their biogeographic distribution suggests that Synechococcus ecotypes exhibit thermal niche preferences. Temperature is a key environmental variable and the elucidation of the temperature stress acclimation in members of this genus can shed light on the molecular mechanisms involved in their adaptive capability. The growth of four representative Synechococcus isolates of various ecotypes from tropical and temperate regions were monitored under various temperature conditions. This revealed drastic differences in growth rates in correlation with their thermal niche preferences. The temperate strains CC9311 and BL107 displayed higher growth rates at lower temperatures while tropical strains WH8102 and WH8109 grew better at higher temperatures. In order to further elucidate their thermal niche preference, the molecular factors influencing the temperature-related growth patterns were explored through global proteomic analysis of WH8102 and BL107. Whole cell lysates of the strains grown at different temperature conditions were fractionated using 1D SDS-PAGE and analysed using label-free quantitative proteomics. Protein identifications provided 27% and 40% coverage of the whole genome for WH8102 and BL107, respectively. Quantitation of protein expression revealed 22% and 20% of the identified proteins were differentially expressed in WH8102 and BL107, respectively. The results were further investigated using qRT-PCR and PAM fluorometry. Differential expression revealed that low temperature appeared to have a significant effect on the photosynthetic machinery. The light harvesting components, phycobilisomes exhibited a reduced expression which could be the result of protein degradation due to photo-oxidative damage and/or as a mechanism to restore the energy balance disturbed as a consequence of low temperature. The lowered phycobilisome expression is found to be a common low temperature-related response between the tropical and temperate isolates. Within the photosynthetic reaction centres, differences in the expression of some core proteins were observed between the two isolates. The expression of core proteins could correlate with the efficiency of repair mechanisms involved in the replacement of photo-damaged core proteins. This differential expression sheds light on the underlying factors which potentially influence the differences in the thermal ranges of tropical and temperate isolates.

 

In September, JAMS was back into top gear, with a bigger audience, and a room with a view. Kent Lim from Macquarie University led off with a talk on his PhD work on the biocontrol agent Pseudomonas strain Pf5. As is often the case in science, things didn’t work out as expected, and Kent found that knocking out suspected pyochelin transporters led to an increase rather than a decrease in efflux of this siderophore and its metabolic precursors. Kent valiantly soldiered on, applying qRT-PCR and Biolog phenotype microarrays to untangle the problem, but unfortunately, this released even more worms from the seemingly-bottomless can provided by strain Pf5. It seems that these transporters may in fact also be regulatory proteins, explaining the unexpected pleiotropic effects of the knockouts.
 

Event Date: 
Wednesday, June 27, 2012 - 18:15 - 18:30
Institution: 
University of Western Sydney
Title: 

The Taguchi methods, or how to quickly and efficiently optimise PCR conditions.

Abstract: 

Originally, the Taguchi methods were formulated for the optimisation of industrial processes, where several factors (3 to 50) of complex multifactorial experiments were tested at different levels (Taguchi, 1986). The Taguchi methods use orthogonal arrays to organise the ‘control’ parameters/factors affecting a process and the levels at which they should vary. A particular algorithm (quadratic loss function) is then applied in order to predict the optimum conditions of a process, whilst accounting for performance variations due to ‘noise’ factors beyond the control of the design. In a normal factorial strategy, every parameter should be individually tested at several levels, thus becoming extremely time-consuming, labour-intensive and expensive. The Taguchi methodology allows for testing only a few combinations, therefore dramatically decreasing the total number of experiments and simultaneously identifying the optimum condition of several factors.
Because some functional genes are present only in small fractions of microbial communities, and only few copies can be present in each genome, their detection by classical PCR methods can be challenging. Optimisation of the experimental conditions of a PCR includes the different components of the reaction mix (concentrations of salt, primers, enzyme, DNA template, etc.) as well as the cycling features (time and temperature of the denaturation, annealing and extension steps, number of cycles, etc.). We used this approach for the optimisation of the detection by PCR of functional genes of non-cultivable microorganisms present in environmental samples. In particular, we tested the different parameters involved in a (touchdown/nested) PCR and estimated the optimum settings for the detection of the functional gene pmoA, coding for the putative active site of the particulate methane monooxygenase, involved in the oxidation of methane by methanotrophic bacteria. The application of the Taguchi method allowed the suppression of a nesting step and thus a significant reduction in the amplification time, as well as reagent cost.
 

Syndicate content