Grant: Research Tools and Instruments - Category 1 (<$150,000)

Funds from this application are sought to purchase a state-of-the-art cell disruption system. The 3 applicants are all microbiologists and routinely grow bacteria such as Escherichia coli as a vehicle to overexpress recombinant proteins or enzymes. In fact, these procedures are the life-blood of the operations of the laboratories involved in the application so the equipment will be a vital component of the infrastructure in the Department of Microbiology & Immunology at Western Ontario. Bacterial cell disruption is an essential step in the preparation of cellular fractions for preparative and analytical studies. The most effective cell disruption systems should (i) permit the solubilization of the maximum amount of the product present in the cell while still maintaining maximum biological activity, (ii) disrupt cells rapidly and to completion to avoid mechanical or artificial alteration of the product such as denaturation and oxidation, and (iii) limit the detrimental effects of the disruption stage on the following separation steps. Traditionally, this is accomplished by high-pressure cell disruption using a French press, which is no longer being manufactured, or by ultrasonic disruption. Both methods have shortcomings for the purification of proteins. French Press is very cumbersome and the cells are heavy and biohazardous material frequently leaks out around the vicinity of the machine. Sonication often prevents protein detection likely due to denaturation induced by heat dissipation during the procedure, and some recombinant cells are resistant to ultrasonic disruption. Many non E. coli species of bacteria, especially S. aureus that is used in the Heinrichs lab, are exceedingly more resistant to breakage by these two methods.