Research Work Summary
Postdoctoral Research Work
The National Food Research Institute at Tsukuba in Japan is heavily involved in the construction of chimeric enzymes. Recent work there suggests that enzyme character can be improved by this method. During my time there I constructed Agrobacterium tumefaciens and Cellvibrio gilvus based chimeric b -glucosidases by shuffling genes in the C and N terminal regions. For example, an Agrobacterium tumefaciens based chimera was constructed by replacing a percentage of the gene in the C-terminal region of Agrobacterium tumefaciens with the corresponding homologous region of Cellvibrio gilvus.
The chimeric genes were cloned in the high expression vector, pET28(a) and were over expressed (approx. 80% of total cellular proteins) in E. coli BL21(DE3). As nearly all target protein was trapped into inclusion bodies much experimentation was performed to solublise this protein in its active form. Some methods used were; in-vivo by co-expression of groEL/ES and in-vitro by rapid dilution and folding in the immobilized state, this involved exposing the inactive proteins to very shallow negative urea gradients.
These Recombinant DNA Technology methods involved basic plasmid manipulation and cloning using current genetic engineering kits, the Polymerase Chain Reaction (PCR), DNA sequencing, scale-up and fermentation, FPLC protein purification and characterization.
Ph.D Research Work
My Ph.D research work involved the design and construction of a whole organism biosensor to monitor pollution in water. The biosensor was based on the respiration rate change in selected microorganisms to pollutant insult.
Soil microflora were screened for maximum response using an oxygen electrode to the following common herbicide pollutants; simazine, chlorotoluron and mecoprop. Low molecular weight ‘redox’ couples was then used as a mediators to monitor electron flow in the selected microorganism by measuring their oxidation at an amperometric transducer. By combining the varying responses of these microorganisms it was possible to determine both the pollutant and its concentration.
The techniques essential to this work can be broken down into the:
Undergraduate Research Work Experience
I was employed for a six-month placement at Boston University School of Medicine, USA. During this period I was trained in the following aspects of blood processing:
However, my main task was to evaluate a new membrane based technology to deglycerolize frozen red blood cells. The membrane was a spinning filter and would allow the processing system to become ‘closed’. This would reduce the risk of contamination and would potentially allow the red blood cells to be processed and stored for greater than 24 hours at 4° C.
Projects undertaken as part of my B.Sc (Hons) Biotechnology Degree
Final year Research Project
The efficiency in degradation of barley b -glucan by yeast secreted b -glucanase
Pauric J. Mc Ginty