Pseudomonas aeruginosa is an opportunistic pathogen, causing a wide range of infections. A major problem in the treatment of Pseudomonas aeruginosa infections is its resistance to antibiotics, so alternative treatment options are being sought. The aim of the master's thesis was to construct a conjugation-based antimicrobial agent that could be used against Pseudomonas aeruginosa. We have chosen the broad-host-range plasmid RK2 that is capable of transmission, replication and maintenance in most genera of Gram negative bacteria. The aim of the thesis was to clone the selection marker gene and the gene for the toxin bacteriocin, colicin E7 into the plasmid. A further objective of the thesis was to evaluate conjugal transfer frequencies of the constructed plasmid from various strains of Escherichia coli to Pseudomonas aeruginosa. The donor of the constructed plasmid, Escherichia coli, is immune to the toxic effect of the protein colicin E7, as it contains the colicin E7 immunity gene. In the recipient cells that do not carry the gene of the immunity protein, the colicin E7 would be lethal. We successfully prepared RK2-Gm plasmid and showed that introduction of the selective marker did not affect the conjugal transfer frequencies. The conjugation frequencies of the RK2-Gm plasmid from the donor strains Nissle 1917, HB101 and DH10B into the recipient strain Pseudomonas aeruginosa L-995 were around 10–6, but around 10–5 from the donor strain SE15. The plasmid RK2-Gm-ColE7a was not successfully prepared however, all the methods used in the thesis are presented.
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