Three mini-projects were completed as part of the MSc course.
The first Miniproject used microarrays to compare the genomes of different types of cyanophage. Cultures of Synechococcus WH7803 were grown in Artificial Sea Water until they were a dark ‘salmon pink’ colour, which took 1-2 weeks. The cultures were then inoculated with phage. When infecting a bacterium, the phage will disable the host gene expression. As a result, the phage which infect cyanobacteria need to have copies of the genes for any proteins used in photosynthesis, to prevent the host from dying before the phage can be reproduced.
If these genes are conserved between different phage, they should be detected by the microarrays. Other such conserved genes should also be detected by this method. This will give an indication of how similar the genomes are, without the expense of obtaining full sequences.
The phage DNA was extracted and purified. The resulting DNA was run on an agarose gel to check for purity, followed by a nanodrop measurement to get the concentration. 1000 nanograms of DNA were required for the microarays. The arrays were read and the data was analysed to determine which genes were common to different types of virus.
The second Miniproject was programming based and was part of a larger project to evaluate methods for detecting Regulatory Modules in DNA.
From the abstract to the thesis:
The number of methods for computationally locating regulatory modules is increasing steadily. Many of these use different principles, such as checking for conserved regions or looking for repeated motifs associated with Transcription Factor Binding Sites. The performance of the different methods is likely to vary according to their nature. Some methods might work well if the species are closely related while others might perform etter for more distantly related ones. Here we present a framework for testing and comparing methods for omputational (or ‘in silico’) detection of Cis-regulatory modules. The framework consists of a small number of computer programs which allow the output of different methods to be compared. Method parameters can be varied and the effect on detection rate can be determined. The results are analysed by determining whether a method correctly identified regulatory module (TP or True Positive) or whether it incorrectly predicted a module where one should not be present (FP or False Positive). The sensitivity nd specificity of the methods can then be determined. The further apart species are in an evolutionary sense, the more different their DNA is. This presents a challenge when using conservation as a basis for locating regulatory modules. While it can sometimes be fairly straightforward to locate regions in closely related species, this not the case in more distant species where the genome can be significantly different. While some regulatory modules have changed very little since mammals and fish shared a distance ancestor, others have appeared, disappeared or changed significantly. The detection rate therefore decreases in line with ‘species distance’.
Imaging Bacterial Nanowires.
There are many dierent types of bacteria which can use both oxygen and metallic cations as the electron acceptors during respiration. One such bacteria is Shewanella oneidensis . Under oxygen poor conditions it favours metal ion reduction using membrane bound cytochromes to perform electron transport. When metal ions are not in direct contact, a `nanowire' containing these cytochromes can be produced which enables the bacteria to scavenge for metals at a greater distance. We investigate dierent methods of imaging and analysing these nanowires.
Shewanella oneidensis is a gram negative bacteria which was originally known as Shewanella putrefaciens and was identied as a contributor to food spoilage. The Shewanella oneidensis MR-1 strain was isolated from Lake Oneida in New York state and was named after its metal reducing ability. It can tolerate variations in acidity (pH range 5.6-9.4), temperature (up to 35 C) and oxygen availability, growing successfully (albeit slowly) at temperatures as low as 3 C. It can use a wide variety of energy sources, mainly simple 2 or 3 carbon organic acids or sugars such as formate, acetate or lactate. When oxygen is scarce it can utilise some metal salts or oxides as electron acceptors.