Konjugacijski prenos plazmida pOX38a v realnem času

magistrsko delo

Bernarda Strojin (Author), David Stopar (Reviewer), Marjanca Starčič Erjavec (Mentor), Natan Osterman (Co-mentor)

Abstract

Večkratno proti antibiotikom odporne bakterije postajajo čedalje večji problem. Geni za odpornost proti antibiotikom se največkrat nahajajo na konjugativnih plazmidih, ki so sposobni horizontalnega prenosa med dvema celicama, ki sta v neposrednem stiku (=konjugacija). Konjugacija je razlog za hitro širjenje odpornosti v bakterijski populaciji. Alternativa antibiotikom bi lahko postali bakteriocini. Cilj magistrskega dela je bil vizualizirati direkten konjugacijski prenos plazmida pOX38a z zapisom za kolicin E7 z mikroskopom, opremljenim z optično pinceto. Ker nam to ni uspelo, smo v nadaljevanju preverjali možne razloge za ta neuspeh. Določali smo frekvenco konjugacijskega prenosa izbranega plazmida ter preverjali vpliv minimalnega gojišča in vpliv prenašanja bakterij na frekvenco konjugacije. Ugotovili smo, da minimalno gojišče in prenašanje bakterij na frekvenco konjugacije ne vplivata. Nadalje smo želeli ugotoviti način delovanja kolicina E7 na recipientski sev. Zanimalo nas je ali celica po delovanju kolicina lizira in če se spremeni prepustnost membrane in omogoči vstop propidijevega jodida. Ugotovili smo, da kolicin E7 v dovolj visoki koncentraciji deluje bakteriocidno na bakterijski sev. To smo dokazali z merjenjem optične gostote pri valovni dolžini 600 nm in določanjem števila CFU/mL. Do lize oziroma spremembe prepustnosti membrane za vstop propidijevega jodida ne pride. Po 24 urah inkubacije bakterijskega seva s kolicinom lahko nekatere celice pridobijo odpornost proti kolicinu E7, kar kaže zvišanje vrednosti optične gostote in števila CFU/mL.

Keywords

Escherichia coli;konjugacija;fluorescenca;konjugativni plazmidi;kolicin E7;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.09 - Master's Thesis
Organization:	UL BF - Biotechnical Faculty
Publisher:	[B. Strojin]
UDC:	579.25:579.842.1/.2:577.2
COBISS:	5099640
Views:	538
Downloads:	201
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Conjugation transfer of pOX38a plasmid in real time
Secondary abstract:	Multidrug resistant bacteria are becoming an increasing problem. Often antibiotic resistance genes are carried on conjugative plasmids i.e. plasmids that are capable of horizontal gene transfer between two bacterial cells in direct contact (=conjugation). Bacteriocins could become an alternative to antibiotics. The aim of this Master Thesis was to visualize the transfer of pOX38a plasmid from the donor to the recipient cell under the microscope, equipped with the optical tweezers. As we did not succeed, we next examined the possible reasons for this failure. We determined the frequency of the pOX38a conjugal transfer and influence of minimal medium and bacterial carriage on conjugation frequency. Our results showed that neither minimal medium nor the bacterial carriage affected the conjugation frequency. A further objective of the Thesis was to determine the effect of colicin E7 on the recipient strain. We were interested, if the recipient cell lysed after addition of colicin E7 to the bacterial culture and if the cell membrane permeability was changed in the way that propidium iodide can enter the cell. We found that a high enough concentration of colicin E7 has a bactericidal activity resulting in decrease of the optical density of the bacterial culture at a wavelength of 600 nm and lower number of CFU/mL. However cells did not lyse and the membrane permeability for propidium iodide was not changed, so propidium iodide did not enter the cells. After 24 hours of incubation with colicin E7 some cells acquired colicin E7 resistance, as optical density and number of CFU/mL increased again.
Secondary keywords:	Escherichia coli;conjugation;fluorescence;conjugative plasmids;colicin E7;
Type (COBISS):	Master's thesis/paper
Study programme:	0
Thesis comment:	Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Pages:	XI, 47 f., [3] f. pril.
ID:	11228344