GENE, ARS-R ANCHORAGE CASSETTE, ARS-R EXPRESSION- ANCHORAGE CASSETTE, RECOMBINANT PLASMID, BACTERIAL TRANSGENIC LINEAGE, USE OF SAID GENE, USE OF SAID LINEAGE IN ENVIRONMENTAL BIOREMEDIATION PROCESSES
World Intellectual Property Organization Patent
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The present invention relates to the construction and insertion of a DNA plasmid vector of broad spectrum for Gram-negative bacteria, that carries a gene sequence which, when expressed, enables the anchorage of a chelating protein for arsenic ions on the Gram-negative bacteria cellular surface. For that end, the structural sequence of the regulatory arsR gene without stop codon (SEQ ID N° 1 ) was amplified by Polymerase Chain Reaction (PCR) using as a template the chromosome 1 of Cupriavidus metallidurans, CH34 lineage and inserted into the pGEM-T cloning vector, yielding the pGEMT-As plasmid (SEQ ID N° 2). The expression vector containing the sequence encoding the cassette for the expression and anchoring of heterologous proteins in Gram-negative bacteria, under the control of the pan promoter (SEQ. ID N° 3), was obtained upon digestion of the pCM-Hg plasmid with Xbal and Sa/I restriction enzymes. The arsR gene was released from the pGEMT-As plasmid by digestion with Xba\\\\ and Sa/I restriction enzymes and then ligated to the linearized expression vector, called pCM (SEQ. ID N° 4), resulting in the construction of the pCM-As plasmid (SEQ ID N° 5). Additionally, the present invention provides recombinant strains of Gram-negative bacteria containing said recombinant plasmid, method of production, use of the recombinant plasmid to enhance bacterial arsenic resistance and capability to adsorb arsenic ions, as well as the use of the transgenic strains for the adsorption of arsenic ions in environmental bioremediation processes, with the possibility of recovering the metalloid as a byproduct.
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