Dr. Vito Guiseppe D’Agostino
Dr Vito is an Associate Professor at the Department of Cellular, Computational and Integrative Biology - CIBIO at the University of Trento, Italy. His research focuses on cell cultures, cellular stress response, epithelial-mesenchymal transition, genetic engineering, molecular biology, motor neuron disease, protein-RNA interaction, reporter gene assay, RNA binding, and RNA translation. We first introduced our project and the methodology to Dr Vito, and upon hearing this, he gave us several suggestions.
He noted that in experiments where we can expect both upregulation and downregulation of the signal depending on the 5’ UTR we use, it is crucial to keep in mind that the relative stability of GFP/RFP is quite high. If the 5’ UTR under consideration is downregulating the protein's translational rate, quantifying this downregulation is a challenge since the fluorescent marker remains in the cell and continues to fluoresce for a prolonged duration due to its high stability. In comparison, 5’ UTR sequences that result in upregulation are easier to characterize. The CDS of a small-sized reporter protein (among the several versions of the fluorescent markers) can be employed as they have lower stability to remove the problems with characterizing the downregulation.
He suggested we use bacterial strains that could be defective in specific ribosomal proteins or the rRNA, as their translational capacity would be much lower than a strain with no defects in the abovementioned parts. As the translation rate would be lower in these strains, it would be easier to characterize the effect of the 5’ UTRs that are responsible for pushing the translational efficiency of the sequence we are using.
He mentioned finding a method to optimize and increase the production of mRNA that preserves not only the bio-utility of E. coli cells but also increases the amount of protein produced in the cells would be very useful for producing biotherapeutics at a lower cost. A case where it will be specifically significant is the production of insulin, a lifesaver hormone for millions of diabetic patients that is still being produced in E. coli. He also said that it is unlikely that a 5’ UTR sequence with promising results in E. coli would show similar results in a mammalian system.
When asked about the sustainability practices we could employ in our experiments, he recommended using glass equipment wherever possible instead of plastic equipment since it is sustainable and easy to autoclave, clean and maintain.
