A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.

A method for transformation of Geobacillus debilis by conjugation was developed using a recombinant plasmid, pNW33N-pxyl-bs2-mob, derived from pNW33N. The plasmid includes the mob region of RP4 for mobilization, is mobilized from E. coli S17-1 to G. debilis, and can stably propagate in G. debilis trans-conjugants grown at 50 oC and 55 oC, in the presence of thiamphenicol. Successful conjugation was depended on the cell density and viability of G. debilis when harvested for conjugation, as well as the metabolic activity of E. coli S17-1 used for conjugation. Substantial reduction in size of the plasmid DNA was observed when G. debilis transconjugants were cultured at 60 oC in the presence of thiamphenicol, and uniform rearrangement of the plasmid DNA was observed after culturing G. debilis transconjugants in the presence of spectinomycin, even at 50 oC.

Thesis (PhD)--University of South Australia, 2001.

Reprints to two articles published by the author held in pocket in back of publication / x, 158, xxvi leaves : ill. ; 26 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1974

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaves 103-113). Also available via the Internet.

Transglutaminases (protein-glutamine:amine y-glutamyl- transferase, EC 2.3.2.13) are a family of calcium-dependent enzymes which catalyze an acyl transfer between glutamine residues and a wide variety of primary amines. When lysine acts as the acyl-acceptor substrate, α-glutamyl lysine isopeptide bond is formed. Isopeptide catalyzation results in protein cross-linkage which is prevalent throughout biological processes. Microbial transglutaminase (mTG) is a bacterial variant of the transglutaminase family, distinct by virtue of its calcium-independent catalysis of the isopeptidic bond. Furthermore, mTGs promiscuity in donor substrate preference highlights its biocatalytic potential. To realize the potential of the enzyme, a high-reactivity tag was necessary for protein labelling. To address this, an enzyme-coupled assay was developed to characterize peptides in the hopes of developing orthogonal substrates to facilitate mTG-mediated labelling and biocatalysis. The discovery of high-reactivity peptide tags allowed the realization of in vitro protein labelling- facilitated by mTG. The 7M48 peptide was fused to a test protein, where it was subsequently propargylated with propargyl amine to fluorescently label or immobilize a test protein. Although there are endless possibilities for in vitro bio-conjugation through mTG, proteolytic activation limits any in-cell labelling strategies with this enzyme. To circumvent this issue, development of an alternative bacterial enzyme, Bacillus subtilis transglutaminase (bTG), was chosen to replace mTG. bTG maintains the advantages associated with mTG but is expressed in its active form. Unlike mTG, there is limited preliminary research associated with the enzyme or its substrate scope. To better understanding substrate reactivity, a FRET-based assay was developed allows for the discovery of new high-reactivity peptides for bTG. These peptides were then used in labelling strategies to demonstrate the potential bTG-mediated bioconjugation. This strategy includes the added advantage of potential for in-cellulo labelling.

The therapy suppressive tumour microenvironment (TME) continues to hinder anti-cancer therapies. Local delivery of therapeutic proteins, including potentially toxic factors, is increasingly needed to enhance immunotherapeutic bioactivities and minimize systemic toxicity. To this end, we are developing vehicles that immobilize to extracellular matrix (ECM) components upregulated in TME for localization of polymer-grafted bioactive cytokines with tunable degradation rates to control cytokine clearance. The grafted cytokine would be bioactive, and the length of the therapy would be governed by the degradation kinetics of the hydrolytic linker between the cytokine and polymer. The cytokines were expressed and purified, and their biological activity was confirmed. Click chemistry was used to graft the therapeutic proteins and collagen-binding peptides to the copolymer. Production of the therapeutic carriers was confirmed by SEC and fluorescent measurements. Biolayer interferometry and tracking immobilization inside collagen gel confirmed the binding affinity between carriers and collagen type 1. In vitro studies confirmed the bioactivity of the carriers in the presence of T-cells and macrophages. In summary, ECM binding vehicles for local sustained protein release will aid in the local delivery of therapeutic proteins to alter TME and promote immunotherapies. Screens will be conducted in multicellular spheroid models to identify bioactive formulations. / Thesis / Master of Science (MSc)

No description available.

No description available.

Tuberculosis (TB) is an important disease worldwide. Currently, one-third of the world’s population is infected with TB, and it is a leading cause of death among people living with HIV. Immediate but also accurate diagnosis is required for disease control, yet available diagnostics cannot do both simultaneously. Therefore, designing a technique that can diagnose the disease correctly in the shortest possible time is in great demand in order to stop its spread. Diffraction-based sensing is a novel technique for measuring of biomolecular interaction that has potential for disease diagnosis. In this study, diffraction-based sensing successfully demonstrated its usefulness for diagnostics of TB using recombinant TB antigen, or by detection of interferon-γ that is produced from white blood cells when the immune system activates. The feasibility of the technology was also evaluated in terms of providing real time observation, reducing diagnostic duration, and increasing sensitivity of detection.