Jo-Anne Chuck

High frequency transformation of the Amphotericin-producing bacterium Streptomyces nodosus

This study has investigated DNA transformation in the Amphotericin-producing organism Streptomyces nodosus. Amphotericin B is an antifungal drug with severe side effects in humans and the availability of structural variants would aid investigations into the mode of action and cytotoxity of the drug. Analogs of related polyketide drugs have been rapidly made by genetic engineering of biosynthetic genes; however, this requires the introduction of foreign DNA into the host. Protocols for protoplast formation and regeneration were established; however, preparations were recalcitrant to DNA uptake. Electroporation-mediated methodologies also were not successful. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies of 5 x 10(-5) exconjugants generated per recipient. Use of DNA methylation-impaired E. coli donor strains resulted in 100-fold higher transformation efficiencies, indicating that DNA methylation recognition systems are operable in the organism. This methodology will enable genetic and biochemical analysis of the gene cluster responsible for making Amphotericin B.

Viability analysis of alginate encapsulated micro-organisms using fluorescent stains

The encapsulation of micro-organisms such as bacteria and fungi in biopolymers is currently being evaluated as delivery systems in many fields. Information about the viability and morphology of the organisms in the microparticle is often required to ascertain the longevity of the systems. A rapid method using fluorescent stains for microbial viability has been validated for organisms within alginate microparticles. Usually viability is assessed by dissolving the microparticles and cell culturing. This new method is advantageous for slow growing or filamentous organisms because these are not quickly or accurately enumerated by plate counts. In addition, the technique also allows the morphology of the organism to be monitored over time.

Hypothetical biotechnology companies: A role‐playing student centered activity for undergraduate science students

Science students leaving undergraduate programs are entering the biotechnology industry where they are presented with issues which require integration of science content. Students find this difficult as through‐out their studies, most content is limited to a single subdiscipline (e.g., biochemistry, immunology). In addition, students need knowledge of the ethical, economic, and legal frame work in which the industry operates. This article presents an approach to deliver these outcomes in a collaborative and active learning modality which promotes deep learning. In the model, groups of final year undergraduate students form hypothetical biotechnology companies and identify real issues of interest to industry, make integrative team decisions, use professional level technology, and develop appropriate communication skills. The final successful teaching paradigm was based on self reflection, observation, and student feedback to ensure appropriate attainment of content, group work skills and increased confidence in professional decision‐making. It is these outcomes which will facilitate life long learning skills, a major outcome applicable for all tertiary education. Biochemistry and Molecular Biology Education Vol. 39, No. 2, pp. 173–179, 2011

The isolation of isoagathenediol: a new tricyclic diterpene from the lipids of Rhodospirillum rubrum

Summary: A tricyclic diterpene, isoagathenediol, has been isolated from the lipid fraction of the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. The compound was present in trace amounts and its structure was determined by NMR spectroscopy and mass spectrometry. Isoagathenediol is a new natural product and may function as a membrane fluidity modulator. Five other bacterial species have also been screened for the presence of this compound, but the diterpene was detected only in extracts derived from R. rubrum. As tricyclic diterpene fossils are readily detected in oil and shale samples, the isolation of isoagathenediol has indicated the biological origin of these fossils.

Amplification of DNA Encoding Entire Type I Polyketide Synthase Domains and Linkers from Streptomyces Species

Polyketides are a group of bioactive compounds from bacteria, plants, and fungi. To increase the availability of analogs for testing, the active sites of polyketide synthases are often substituted with homologous domains having altered substrate specificities. This study reports the design of polymerase chain reaction primers that enables isolation of entire active site domains from type I polyketide synthases with native interdomain linkers. This bypasses the need for further genetic screening to obtain functional units for use in genetic engineering. This is especially important in bioprospecting projects exploring new environments for bioresources.

The use of biotin to demonstrate immunohistochemistry, western blotting, and dot blots in university practical classes

Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for detection. This negates the need for a specific primary antibody, saving cost and time. In addition, the easily available and safe reagents allow the methods to be readily adopted without specialist technical expertise. As a result, staff can confidently transfer ownership of the task to the student so as to also develop scientific inquiry skills which promotes student motivation and engagement.

Community structure and antibiotic production of Streptomyces nodosus bioreactors cultured in liquid environments

Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free‐dwelling population. These organisms sporulated with reduced antibiotic production compared with free‐dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid–liquid interface.

Determining if active learning through a formative assessment process translates to better performance in summative assessment

Formative assessment used in a level 2 unit, Immunology, gave outcomes that were both surprising and applicable across disciplines. Four formative tests were given and reviewed during class time. The students’ attitudes to formative assessment were evaluated using questionnaires and its effectiveness in closing the gap was measured by the students’ final exam performance. Despite general enthusiasm for formative tests, the final exam performances were disappointing. Lessons learnt indicated that the students answered questions with ‘knowledge dumps’ rather than addressing the verb, and that feedback from the instructors, although providing answers, did not empower students to understand the verb in the question. Therefore, using formative tests to identify and address bad habits, rather than providing exam practice, would be a more judicious approach to formative testing and likely to improve the overall skills base of students commensurate with higher learning.

Using Biotinylated Proteins to Demonstrate Immunodetection of Antigens via Western Blotting, Dot Blots, and Immunohistochemistry

Using biotinylated targets for detection by enzyme-linked avidin allows immunodetection methods to become more economic in cost and time as it negates the need for a specific primary antibody. Methods are described to use exogenously added biotin to complex biological samples to demonstrate western blotting, dot blots, and immunohistochemistry. These methods can be used in biological science tertiary teaching laboratories to allow novices to gain skills in a risk-free environment to promote student motivation and engagement.

Education of undergraduate Animal Science and Zoology students in professional practice : can we ensure ethical compliance and educational outcomes?

This case study describes the conflicts between the needs of a university animal ethics committee to meet legislative requirements and the pedagogical outcomes for third year undergraduate project students working with animals in an Australian context. The projects undertaken by this cohort are conducted in collaboration with a wide variety of external clients, using a diverse array of vertebrate species and involve many levels of ‘invasiveness’ or risk. Conflicts are discussed from the perspective of the various parties involved including the committee, academic staff in the role of supervisors, students and external clients. Ways forward are suggested to improve the process but it is clear that education of the committee, academic staff, students and clients is required and that it is essential to ensure open-channels for dialogue and discussion are utilised effectively.