Riassunto analitico
This thesis explores the design of a plant-based expression system in the production of recombinant proteins using Nicotiana benthamiana, with potential applications in sustainable biotechnology. The study focuses on the expression of Cutin Synthase 1 (CUS1), an enzyme that catalyzes the cutin cuticle biosynthesis process, and the implementation of the sacB gene as a counterselection system to facilitate molecular cloning.
To achieve this, expression vectors were designed using the spectinomycin-resistant pTRA backbone, with different signal peptides to study their influence on protein localization and expression efficiency. The constructs were introduced into Rhizobium radiobacter and utilized for transient expression in N. benthamiana. CUS1 expression was evaluated through dot blot analysis, and statistical modeling was applied to identify optimal experimental conditions. Computational analyses, including AlphaFold-based structure prediction and sequence optimization, were also performed to study and comprehend CUS1's characteristics.
Furthermore, the functionality of sacB as a counter selection marker was assessed in R. radiobacter by taking advantage of its toxicity in the presence of sucrose. While initial results confirmed its effectiveness, later experiments showed some divergence from this expectation, which is indicative of something not well understood.
This discovery advances understanding of some of the factors altering plant-made protein expression, especially the importance of signal peptides in protein targeting and stability. The results will contribute to optimizing transient expression systems in plants with possible applications in synthetic biology and sustainable materials production.
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