Optimization of Agroinfiltration Technique for Protein Production in Nicotiana Benthamiana

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[ES] Plants are considered a good system for expression which allows them to be used as biofactories. The use of plants as biofactories has important advantages compared to other techniques used, such as: - Lower cost of production - Obtaining functional proteins free of contaminations of animal pathogens - Safety, not being the stable transformed plant - Ease of purification of obtained proteins - Easy scalability of production Currently the molecular pharming can be considered an option to produce drugs for de In line with the multiple advantages, the proposed Master’s End Work tries to, altering different variables that can influence the expression and production of proteins in plants, increase the efficiency, in order to achieve a high production scalability of the process. For the use of plants as a biofactorate the transient expression is used, a faster strategy than the nuclear transformation of the plant and very efficient for the production of recombinant proteins. Transient expression requires the use of viral vectors and the technique called agroinfiltration. Agroinfiltration consists of the infiltration of Agrobacterium strains using a plasmid containing the genes that are to be expressed in the plant, which allows the transgene to be inserted into the cell nucleus, but does not integrate into the genetic material of the plant. On the other hand, the viral vector allows the rapid infection of the plant. General agroinfiltration is carried out by vacuum so that the agroinfiltration solution penetrates rapidly into the plant tissue. Other techniques for transient expression mediated by Agrobacterium such as spray or immersion are used to test the efficiency of transient expression without the need to vacuum. On the other hand, obtaining good results in terms of expression and production requires certain conditions of OD (bacterial optical density), the course of a certain period of time, certain concentrations of the surfactant used, etc. Different alternative tools to classical agroinfiltration, such as spraying with different instruments, surfactants or immersion (without doing vacuum), are used in this proposal of Master’s Work. The efficiency of transient expression with different ODs is tested, to check to which OD concentration is optimal, with the minimum product investment. Different concentrations of a surfactant product are used (to facilitate the medium penetrates the tissue of the plant), as well as different brands of the product, to obtain, as with the OD, the optimal concentrations for a good efficiency in the expression. On the other hand, the usefulness of submerging the plants already submerged, to increase the expression of the protein of interest in them, is analyzed. The aim is to know the optimal concentrations of OD, surfactant as well as time. This allows to be able to use the right quantity of product without squandering in bacterial crops, which implies the saving of culture media, antibiotics, electricity, as well as surfactant and other resources used. On the other hand, to measure the expression, photography has been used, for it will develop image taking protocols and vegetal area counting protocols infected with programs of wide use such as Photoshop, Image j or Fiji.

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Polytechnic University of Valencia

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Polytechnic University of Valencia

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