Postdoctoral Research Work

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:

Isolation, screening, characterisation and identification of microorganisms showing interesting reactions to the herbicides,
Scanning Electron Microscopy of the isolated bacteria,
design and construction of a bioelectrochemical cell to monitor the electron flow in these bacteria, and
use of cyclic voltammetry and a knowledge of electrode electrochemistry and A.C. Impedance to optimise the bioelectrochemical cell constructed.

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:

to freeze human and baboon red blood cells with 40% W/V glycerol for storage at -80° C and subsequently to thaw and deglycerolize the red blood cells for in vitro studies,

to separate blood into its main components,

to perform complete analysis of whole blood, including lipid identification and

to document the in vitro recovery of red blood cells following processing procedures

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

Literature Surveys:

Biochemical Action of Anti-Depressant Drugs,

Food Additives, and

b -Glucans and b -Glucanases

Pauric J. Mc Ginty