Biología Molecular de Bacterias Gram-positivas

The objective of the training project is to build a plasmid vector for the cloning and inducible expression of genes in the lactic acid bacterium Lactiplantibacillus plantarum. Such a vector will be based on a high-copy-number mutant replicon derived from the lactococcal plasmid pSH71, which replicates by a "rolling circle" mechanism and has a broad host range. The vector will also contain the inducible expression system (derived from the streptococcal maltosaccharide utilization regulon) consisting of the PM promoter/operator and the gene encoding the MalR repressor, which, in the absence of maltose, represses transcription from PM. A multicloning site will be inserted 3' from the PM promoter/operator in order to facilitate the cloning of the genes of interest whose expression in L. plantarum is intended. The functionality of the cloning and inducible gene expression vector in L. plantarum will be tested by cloning the gene encoding the mCherry red fluorescent protein under the control of the PM promoter/operator and quantifying the fluorescence emitted by the host bacteria grown in the presence or absence of maltose. Once the functionality of the inducible expression vector has been validated, the ribU gene of L. plantarum, which encodes the riboflavin transporter, will be cloned. Thus, the successful development of the proposed project will not only lead to the construction of a vector for cloning and regulated gene expression in L. plantarum, but will also allow future studies on the effect of the presence and/or overproduction of the RibU transporter in the production of riboflavin and in the robustness of the recombinant strains.