WP_036402927.1 cianobakterije Microcystis aeruginosa PCC 7806

diplomsko delo

Andraž Rotar (Author), Marko Dolinar (Mentor), Petra Tavčar Verdev (Co-mentor)

Abstract

Sistemi toksin-antitoksin (TAS) so pogosti genski elementi prokariontskih organizmov. Sestavljeni so iz zapisa za toksin, ki v izraženi proteinski obliki onemogoča rast organizma, ter antitoksina, ki ta toksin onesposobi. TAS so na podlagi bioinformacijskih analiz prisotni tudi v genomu cianobakterije Microcystis aeruginosa PCC 7806, ki je ekološko poznana kot povzročiteljica cvetenja sladkih vod in, v določenih razmerah, izloča toksin mikrocistin, ki je nevaren za ljudi in živali. Kljub pomembnosti cianobakterije M. aeruginosa v ekološkem smislu so njeni TAS slabo raziskani in večina med njimi ostaja eksperimentalno nepotrjenih. V sklopu diplomskega dela smo želeli molekulsko klonirati in v bakteriji Escherichia coli izraziti tri zapise za pare toksin-antitoksin (TA). Na podlagi bioinformacijske analize smo izbrali naslednje kandidate: BH695_3840 (toksin) - BH695_3841 (antitoksin), WP_036399356.1 (toksin) - BH695_4980 (antitoksin), WP_002748554.1 (toksin) - WP_036402927.1 (antitoksin). Iz genomske DNA smo z metodo PCR pomnožili in nato izolirali zapise za navedene TA, ki smo jih vstavili v klonirne vektorje, s katerimi smo transformirali bakterije E. coli. Bakterije smo namnožili in iz njih izolirali klonirne vektorje ter z restrikcijskimi endonukleazami zapise izrezali. Novonastale zapise za pare TA smo nato vstavili v ekspresijske vektorje. S končnimi konstrukti smo izvedli testno izražanje v transformiranih bakterijah E. coli. Izraziti nam je uspelo le protein BH695_3841, saj smo naredili napako pri načrtovanju začetnih nukleotidov za ostale konstrukte.

Keywords

cianobakterije;Microcysitis aeruginosa PCC 7806;sistemi toksin-antitoksin;diplomska dela;

Data

Language:	Slovenian
Year of publishing:	2025
Typology:	2.11 - Undergraduate Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[A. Rotar]
UDC:	577:582.232:602.7(043.2)
COBISS:	237788419
Views:	22
Downloads:	4
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Molecular cloning of toxin-antitoxin pairs BH695_3840/3841, WP_036399356.1/BH695_4980, and WP_002748554.1/WP_036402927.1 in the cyanobacterium Microcystis aeruginosa PCC 7806
Secondary abstract:	Toxin-antitoxin systems (TAS) are common genetic elements in prokaryotic organisms. They consist of a toxin-encoding gene, which in its expressed protein form inhibits bacterial growth, and an antitoxin, which neutralizes the toxin. Based on bioinformatic analyses, TAS are also present in the genome of the cyanobacterium Microcystis aeruginosa PCC 7806, an organism ecologically recognized as a contributor to freshwater blooms and, under certain conditions, a producer of the toxin microcystin, which poses a threat to humans and animals. Despite the ecological significance of M. aeruginosa, its TAS remain poorly studied, with the majority remaining unconfirmed experimentally. In this work, we aimed to molecularly clone and express three toxin-antitoxin (TA) gene pairs in Escherichia coli. Based on a bioinformatic analysis, we selected the following candidates: BH695_3840 (toxin) - BH695_3841 (antitoxin), WP_036399356.1 (toxin) - BH695_4980 (antitoxin), and WP_002748554.1 (toxin) - WP_036402927.1 (antitoxin). Using genomic DNA, we amplified the selected TA gene pairs via PCR, isolated them, and inserted them into cloning vectors, which were subsequently used to transform E. coli. The transformed bacteria were propagated, and cloning vectors were isolated and subjected to restriction endonuclease digestion to obtain the TA gene pairs. The resulting cloning regions were then inserted into expression vectors. Finally, test expression of the constructs was performed in transformed E. coli cells. Only the BH695_3841 protein was successfully expressed, due to an error in the design of the initial nucleotides for the other constructs.
Secondary keywords:	cyanobacteria;Microcysitis aeruginosa PCC 7806;toxin-antitoxin systems;Molekularno kloniranje;Univerzitetna in visokošolska dela;
Type (COBISS):	Bachelor thesis/paper
Study programme:	1000371
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, UNI Biokemija
Pages:	1 spletni vir (1 datoteka PDF (39 str.))
ID:	26465198