My project was centred on previous work by Sai Priya Sarma Kandanur in her thesis on the E. coli adhesin intimin, an inverse autotransporter protein, located in the bacterial outer membrane. Intimin is an important virulence factor in bacteria such as Enteropathogenic E. coli (EPEC). It is involved in the formation of attaching and effacing lesions which allow tight adhesion of the bacteria to intestinal epithelial cells. The translocated intimin receptor (Tir) is delivered to host cells via a type 3 secretion system (T3SS).
My role was to produce intimin mutants via transformation of pET22-based plasmids with cloned intimin variants into lab E. coli and induce overproduction of outer membrane (OM) proteins for isolation. The plasmids were transformed into the bacterial cells through a combination of chemical treatment and heat shock, then grown overnight. Plasmid containing colonies were used to inoculate liquid auto-induction media containing glucose and lactose and again incubated overnight. Cells grew to a high density using the glucose and once depleted, the cells utilized the glycerol and lactose. The plasmids contain a T7 promoter that is indirectly activated by lactose leading to the overexpression of intimin. The cells were then harvested via centrifugation. The cells were then lysed in tubes containing glass beads, DNAse and lysozyme using a speed shaker. After the cell debris was removed by centrifugation, the supernatant containing the OMs was collected, washed with HEPES and the inner membranes selectively solubilised using sarkosyl (SLS) treatment. The OMs were washed again, pelleted and finally resuspended in a small amount of HEPES and SDS-PAGE sample buffer ready to be run through an SDS-PAGE gel. Once the bacterial outer membranes were isolated, they were used to produce Western blot images demonstrating the heat shift patterns between heated and unheated samples. The overall aim is for these images to be included in publication of the research. Obtaining satisfactory heat shift images had previously proved difficult. The purpose of these images was to demonstrate the presence and the correct folding of the mutated proteins. The denatured proteins travel slower through an SDS-PAGE gel than correctly folded proteins. The gels were run on ice (see picture) to avoid denaturation of the proteins during the run to ensuring a clearer heat-shift image.
I found the transition from attending teaching labs, in which protocols are largely set up for you, to working in a research lab challenging at first. However, my supervisor was very patient and allowed me to be independent while also being available to answer questions and help me troubleshoot any mistakes! My confidence increased a great deal over the course of the project as I became used to working independently. I came to appreciate how difficult production of good Western blot images can be, but with practice I managed to produce some clear images with visible heat-shift for some of the mutants. I did not manage to get all of the images in the eight-week timespan, but have hopefully provided my supervisor with enough OM preparations to complete the rest. I enjoyed the experience very much and feel it has prepared me well for my upcoming Masters project as well as giving me valuable insight into the nature of research work.
Nottingham Trent University