Venetia A. Saunders

Assessing the Potential of Bacterial DNA Profiling for Forensic Soil Comparisons

ABSTRACT: A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.

Evaluating Employability Skills: Employer and Student Perceptions

Abstract Graduate employability is a key issue for Higher Education. In this two-part study student employability skills have been evaluated from the perspective of sandwich students and graduates in biomolecular science, and their employers. A strong correlation was found between employer and sandwich student/graduate perceptions of the relative priorities amongst employability skills. Skills such as enthusiasm, dependability and team-working scored higher than subject knowledge skills, whilst commercial awareness, negotiation and networking were given lowest priority. Furthermore, the lowest ranked skills were those that sandwich students/graduates were assessed to be least proficient in. Overall skills of new graduate employees were rated less highly by their employers than by the graduates themselves. In the second part of the study an employability skills profile was compiled and distributed to biomolecular science students at levels 1, 2 and 3, as part of personal development planning. Level 3 students rated themselves more highly than level 1 and level 2 students in subject knowledge, most core skills and personal qualities, except tolerance to stress. Implications of this study, including the value of student self-assessment of their skills and utility of the profile to underpin personal development planning and inform graduate recruitment processes, are discussed and recommendations made.

4 Bacterial Transformation with Plasmid DNA

Publisher Summary In general, bacteria that can be transformed by chromosomal DNA can also be transformed by plasmid DNA. However, there are notable differences in the efficiency with which plasmid and chromosomal markers transform competent cells, largely because of the differential processing of various topological forms of DNA. In naturally competent cells, a single plasmid molecule generally requires interaction with another replicon for successful establishment of the transforming plasmid. However, it should be appreciated that transformation in different bacterial species may involve various mechanisms with different requirements for the physiological and genetic state of the recipient and for the topological nature of the input DNA. Transformation can be divided broadly into three stages: binding of DNA to the outside of the cell, transport of DNA across the cell envelope, and establishment of the transforming DNA either as a replicon itself or by recombination with a resident replicon. Transformation systems generally can be saturated by high concentrations of DNA.

Rule-based simulation of temperate bacteriophage infection: Restriction–modification as a limiter to infection in bacterial populations

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria. Such biologically realistic simulations validate the use of the IbM approach to predicting behaviour of bacteriophages in bacterial populations. The applicability of the model for more complex scenarios aimed at predictive modelling of bacterial evolution in a changing environment and the implications for the spread of viruses in a wider context are discussed.

Effects of elevated atmospheric CO2 on fungal competition and decomposition of Fraxinus excelsior litter in laboratory microcosms.

Evidence that chemical changes in litter exposed to elevated CO 2 might alter the composition and function of fungal communities in soil is presented. Some potential effects of elevated atmospheric CO 2 on the decomposition of Fraxinus excelsior leaf litter and the interactions of the colonizing fungi, growing singly or in simple associations in microcosms, were investigated. Fungal colonization was monitored by analysis of the ergosterol content of litter and specific PCR-amplified ribosomal DNA spacer products. After 2 (600 ppm). After triple-species inoculation percentage α-cellulose and, in some combinations, nitrogen content was reduced on litter from seedlings grown in elevated CO 2 .

Rule-based computing system for microbial interactions and communications : Evolution in virtual bacterial populations

We have developed a novel rule-based computing system of microbial interactions and communications, referred to as COSMIC-Rules, for simulating evolutionary processes within populations of virtual bacteria. The model incorporates three levels: the bacterial genome, the bacterial cell and an environment inhabited by such cells. The virtual environment in COSMIC-Rules can contain multiple substances, whose relative toxicity or nutrient status is specified by the genome of the bacterium. Each substance may be distributed uniformly or in a user-defined manner. The organisms in COSMIC-Rules possess individually-defined physical locations, size, cell division status and genomes. Genes and/or gene systems are represented by abstractions that may summate sometimes complex phenotypes. Central to COSMIC-Rules is a simplified representation of bacterial species, each containing a functional genome including, where desired, extrachromosomal elements such as plasmids and/or bacteriophages. A widely applicable computer representation of biological recognition systems based on bit string matching is essential to the model. This representation permits, for example, the modelling of protein-protein interactions, receptor-ligand interactions and DNA-DNA transactions. COSMIC-Rules is intended to inform studies on bacterial adaptation and evolution, and to predict behaviour of populations of pathogenic bacteria and their viruses. The framework is constructed for parallel execution across a large number of machines and efficiently utilises a 64 processor development cluster. It will run on any Grid system and has successfully tested simulations with millions of bacteria, of multiple species and utilising multiple substrates. The model may be used for large-scale simulations where a genealogical record for individual organisms is required.

1 Introduction of DNA into Bacteria

Publisher Summary The introduction of purified DNA into bacteria is an almost universally applicable means of transferring genes between organisms, and has the major advantage that no vector is required: the nucleic acid itself mediates information transfer. The term transformation was originally applied to gene transfer between bacteria that did not require cell-to-cell contact (which distinguishes it from conjugation) and that was sensitive to deoxyribonuclease (which distinguishes it from transduction). The possibilities for introducing DNA into bacteria are numerous. Although various transformation systems are potentially available, there is no doubt that electroporation provides the most widely applicable approach to manipulating previously unexplored organisms. The method is more robust and less subject to the vagaries of cell wall chemistry and nucleic acid metabolism. On balance, electroporation is currently therefore the best place to start when attempting to introduce DNA into a desired organism where there is no existing precedent.

Spatial Variation in Bacterial DNA Profiles for Forensic Soil Comparisons

ABSTRACT The feasibility of using bacterial DNA profiling of soil in forensic investigations is evaluated. Terminal restriction fragment (TRF) analysis of 16S ribosomal DNA may be employed to generate DNA profiles for use to discriminate between soil samples. To assess the localisation of the profiles and strengthen any evidential value of TRF analysis, small-scale spatial variability has been examined. Soil samples were collected at intervals along 2 m horizontal transects of three different ecosystems—a field, a forest, and a dune system—and TRF profiles compared. 13% to 20% of TRFs were common to all sampling points, for all transects. The mean similarity for TRF profiles at all sampling points ranged from 49.3% for the dune system to 58.4% for the field transect. ‘Patch size’ (distance along the transect in which DNA profiles showed highest similarity) ranged from 20–80 cm for each ecosystem. This small- scale variability may cause problems if forensic ‘matching’ of soil DNA profiles is attempted, unless the precise location from which the soil sample was originally taken could be identified. The implications of these findings for forensic fingerprinting of soil samples are discussed.

Factors Affecting Student Choice of the Undergraduate Research Project: Staff and Student Perceptions.

Abstract As pressures on resources are growing and some question the value and types of final year research work for students in the biosciences and other disciplines, it is important to be well informed about student expectations of their project. In this case study within Biomolecular Sciences, questionnaires were used to compare staff and student perceptions about the factors influencing choice of research project by final year undergraduate students. As allocation method changed from choice of specific, predetermined title to negotiation within a wider subject area, any changes in influential factors were investigated. Students on 8 programmes, including joint Honours, were included in the surveys, and 84 responses were analysed, together with those of 20 academics. Results show that whilst staff are aware of most of the factors that influence students’ choice of project, there are differences in some aspects - for example, those relating to the tutor’s role and to the desire for challenging work. Whilst students were satisfied with both methods of project allocation, staff strongly preferred negotiation between staff and students to allocation by specific title. These findings may be useful to others currently reviewing their own practice with regard to project allocation and design.

Chitinase activities from yeast and hyphal cells of Candida albicans

Soluble chitinase activity was purified from yeast and hyphal cells of Candida albicans following ammonium sulphate fractionation, gel-filtration and anion-exchange chromatography. Activity from both cell types resolved into three peaks with identical Mr of 79, 58 and 33 kDa. In both yeast and hyphae the 33 kDa chitinase was the predominant activity. Differences were observed in the relative distribution of the three chitinases in yeast and hyphae and in their substrate specificities. The 33 kDa protein showed preference for the longer chain length glycoside substrates suggesting (endo-) chitinase activity. In contrast, the 79 kDa protein exhibited activity against short and long chain glycosides, suggesting an agglomeration of chitinase and N-acetylglucosaminidase activities. Three separate activities were resolved by gel-filtration but after non-denaturing PAGE a number of bands exhibiting chitinase activity was detected indicating the presence of a complex chitinase system in Candida albicans.