Konstrukcija plazmida RK2-Gm-ColE7a in frekvenca konjugacije pripravljenega plazmida v bakterijo Pseudomonas aeruginosa

Kužnik, Beti (author), Starčič Erjavec, Marjanca (mentor)

Bakterija Pseudomonas aeruginosa je oportunističen patogen, ki povzroča širok razpon akutnih in kroničnih okužb. Velik problem pri zdravljenju okužb z bakterijo Pseudomonas aeruginosa je njena odpornost proti antibiotikom, zato se iščejo alternativne možnosti zdravljenja. Cilj magistrske naloge je bila priprava, na konjugaciji temelječega, protimikrobnega dejavnika, ki bi ga lahko uporabili proti bakteriji Pseudomonas aeruginosa. Odločili smo se za plazmid RK2, ki je plazmid širokega spektra in je sposoben prenosa, replikacije in vzdrževanja v večini rodov, po Gramu negativnih bakterij. Plazmidu smo želeli dodati selekcijski označevalec, rezistenco proti gentamicinu in gen z zapisom za sintezo toksina-bakteriocina kolicina E7 ter ga transformirati v bakterijo Escherichia coli. Nadaljnji cilj naloge je bila ocena frekvence konjugacije konstruiranega plazmida iz različnih sevov Escherichia coli v Pseudomonas aeruginosa. Bakterija Escerichia coli je kot producentka kolicina E7 imuna na toksično delovanje tega proteina zaradi gena imunosti na plazmidu pUC19i oziroma integracije gena za imunost v kromosom. Predvidevali smo, da se bo kolicin E7 v recipientski celici, ki nima zapisa za protein imunosti, izrazil in deloval toksično. Uspeli smo pripraviti plazmid RK2-Gm in s preverjanjem konjugacije potrditi, da pravilen vnos selekcijskega označevalca ne vpliva na frekvenco konjugacije. Frekvence konjugacije plazmida RK2-Gm iz donorksih sevov Nissle 1917, HB101 in DH10B v recipientski sev Pseudomonas aeruginosa L-995 so bile okoli 10–6, iz donorskega seva SE15 pa okoli 10–5. Plazmida RK2-Gm-ColE7a tekom magistrske naloge nismo uspeli pripraviti, predstavili pa smo vse metode, ki smo jih pri tem uporabili.

• Language: Slovenian
• Keywords: bakterije, Pseudomonas aeruginosa, Escherichia coli, plazmidi, protimikrobno delovanje, bacteriocini, kolicin, kolicin E7
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [B. Kužnik]
• Year: 2017
• PID: 20.500.12556/RUL-97797
• UDC: 579.25:577.2:615.33
• COBISS.SI-ID: 4856696
• Publication date in RUL: 12.11.2017

KUŽNIK, Beti, 2017, Konstrukcija plazmida RK2-Gm-ColE7a in frekvenca konjugacije pripravljenega plazmida v bakterijo Pseudomonas aeruginosa [online]. Master’s thesis. B. Kužnik. [Accessed 2 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=97797

Secondary language

• Language: English
• Title: Construction of plasmid RK2-Gm-ColE7a and conjugal frequency of the prepared plasmid in bacteria Pseudomonas aeruginosa

Abstract

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.

• Keywords: bacteria, Pseudomonas aeruginosa, Escherichia coli, plasmids, antimicrobial activity, bacteriocins, colicins, colicin E7