Poskus izolacije cianofaga iz Blejskega jezera v času množičnega pojavljanja cianobakterije Planktothrix rubescens

Andoljšek, Maša (author), Dolinar, Marko (mentor)

Na Blejskem jezeru vsakoletno prihaja do cvetenja cianobakterije Planktothrix rubescens. Možnih vzrokov za hitro zmanjšanje koncentracije te cianobakterije po zaključku cvetenja je več; po eni izmed hipotez je vzrok prisotnost cianofaga, ki napade to cianobakterijo. Vzorčili smo površinsko vodo Blejskega jezera od decembra 2020 do aprila 2021, pri čemer smo poskušali zajeti čim večje število alg. Na osnovi genoma edinega cianofaga, katerega genomsko zaporedje poznamo in napade rod Planktothrix, smo pripravili oligonukleotide, ki bi lahko z reakcijo PCR dokazovali prisotnost sorodnega cianofaga v Blejskem jezeru. Prisotnost Planktothrix rubescens v vzorcih smo dokazali z opazovanjem pod mikroskopom. Poskusili smo izolirati posamezen filament cianobakterije z metodo premikanja proti svetlobi, nato pa še z izolacijo posamezne niti v mikrocentrifugirkah. Opazili smo, da se filamenti orientirajo proti svetlobi. Kulturo Planktothrix rubescens smo poskušali vzgojiti v tekočem gojišču BG-11. Z večstopenjsko filtracijo s končno velikostjo por 0,2 μm smo želeli priti do frakcije z obogatenim fagom. Posneli smo UV-VIS spektre in opazovali, kako so se zmanjšale kontaminante med filtracijo. Z metodama verižne reakcije s polimerazo in agarozne gelske elektroforeze smo v okoljskem vzorcu in končnemu filtratu dokazali, da so v vzorcih prisotne cianobakterije. Poleg tega smo z uporabo oligonukleotidov, na osnovi genomskega zaporedja za Planktothrix specifičnega faga, v istih vzorcih dobili pozitiven rezultat pri dokazovanju prisotnosti cianofagov.

• Language: Slovenian
• Keywords: Planktothrix rubescens, cianobakterija, cianofag, cvetenje, Blejsko jezero
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-137147
• COBISS.SI-ID: 110383107
• Publication date in RUL: 02.06.2022

Secondary language

• Language: English
• Title: Experiments for isolation of a cyanophage from Bled Lake in the period of mass occurrence of the cyanobacterium Planktothrix rubescens

Abstract

Lake Bled is home to an annual bloom of the cyanobacterium Planktothrix rubescens. There are several causes for the rapid decrease in cyanobacterial concentrations as the bloom ends, one hypothesis is the presence of cyanophage which infects cyanobacteria. We sampled the surface water from December 2020 to April 2021, trying to capture as much algal mass as possible. Based on the genome of the only sequenced cyanophage which attacks the genus Planktothrix we designed oligonucleotides that could provide evidence for the presence of a related cyanophage in Lake Bled. The presence of Planktothrix rubescens was proven by microscopic observation. We tried to isolate a single filament of cyanobacteria on the basis of phototropism and by isolating single strands in microcentrifuge tubes. We tried to culture Planktothrix rubescens in BG-11 liquid medium and regularly monitored the turbidity. Multi-stage filtration with a final pore size of 0.2 μm was used to obtain a phage-enriched fraction. We determined UV-VIS spectra and observed how contaminants were reduced during filtration. We have proven the presence of cyanobacterial DNA in our environmental samples and in the final filtrate by polymerase chain reaction and agarose gel. In addition, using our oligonucleotides, we detected presence of cyanophage DNA in the same samples.

• Keywords: Planktothrix rubescens, cyanobacterium, cyanophage, algal bloom, Lake Bled