20.500.12556/RUL-129828 Bioinformacijska analiza parov toksin-antitoksin cianobakterije Microcystis aeruginosa PCC 7806 in molekulsko kloniranje antitoksinov Bh695_0426 in Bh695_0430 Bioinformatic analysis of toxin-antitoxin pairs in the cyanobacterium Microcystis aeruginosa PCC 7806 and molecular cloning of antitoxins Bh695_0426 and Bh695_0430 Sistemi toksin-antitoksin (TA) so genetski elementi, ki zapisujejo za relativno stabilen proteinski toksin, ki onemogoči rast celice, in nestabilen antitoksin, ki zavre škodljivi učinek kompatibilnega toksina. Njihova biološka vloga še ni povsem razumljena, znano pa je, da sodelujejo pri mnogih bioloških procesih, izmed katerih izstopajo postsegregacijska smrt, abortivna infekcija in bakterijska persistenca. Najdemo jih na številnih plazmidih in kromosomih mnogih arhej ter bakterij, med katere sodi tudi cianobakterija Microcystis aeruginosa PCC 7806. Kljub številčnosti sistemov TA v genomu te cianobakterije jih je bilo do sedaj le nekaj tudi eksperimentalno ovrednotenih, zato smo v sklopu tega diplomskega dela želeli zapisa za dva antitoksina izraziti in izolirati iz celic Escherichia coli. Z bioinformacijskimi orodji smo zbrali in analizirali predvidene pare TA ter izmed teh izbrali dva, katerih zapisa za antitoksina smo pomnožili. Z metodami molekulskega kloniranja smo gena za antitoksina Bh695_0426 in Bh695_0430 vstavili v klonirni vektor pJET1.2/blunt. Prenos v ekspresijski vektor pET-28b(+) ni bil uspešen. Uspešno izražanje genov za antitoksina in izolacija proteinskih produktov bi omogočila nadaljnje raziskave njihove strukture in funkcije, pa tudi regulacije izražanja TA. Zaradi tega bi bilo smiselno eksperimentalno delo z manjšimi spremembami ponoviti. Toxin-antitoxin (TA) systems are genetic elements that encode a relatively stable protein toxin that inhibits cell growth and an unstable antitoxin that inhibits the deleterious effect of the cognate toxin. Their biological function is not yet fully understood. However, they are known to participate in many biological processes, most important being postsegregational killing, abortive infection and bacterial persistence. They can be found on plasmids as well as chromosomes of numerous archaea and bacteria, including cyanobacterium Microcystis aeruginosa PPC 7806. Despite the abundance of TA systems in the genome of this cyanobacterium, only a few have been experimentally characterised so far. Therefore, our goal has been to express two antitoxins and isolate the protein products from Escherichia coli cells. Using bioinformatics tools, we collected and analysed the predicted TA pairs. From these, we selected two and amplified their antitoxin sequences. Using molecular cloning methods, we inserted the genes for the antitoxins Bh695_0426 and Bh695_0430 into the cloning vector pJET1.2/blunt. However, transfer of the antitoxins into the expression vector pET-28b(+) was unsuccessful. Successful expression of antitoxin genes and the isolation of protein products would enable further research of antitoxin function, structure and regulation of TA gene expression. It would therefore be reasonable to repeat experiments with a few adjustments. cianobakterije Microcystis aeruginosa PCC 7806 sistemi toksin-antitoksin antitoksin cyanobacteria Microcystis aeruginosa PCC 7806 toxin-antitoxin systems antitoxin true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2021-09-08 14:40:05 2021-09-08 14:40:10 2022-09-09 04:13:28 0000-00-00 00:00:00 2021 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 15862 80181251 Glavic_Kim_-_Bioinformacijska_analiza_parov_toksin-antitoksin_cianobakterije_Microcystis_aerugin.pdf Glavic_Kim_-_Bioinformacijska_analiza_parov_toksin-antitoksin_cianobakterije_Microcystis_aerugin.pdf 1 698CF05ABF5F6D17A6B3142D828B1773 044e4f440df4e67744d44de0715031a5b69850aa1bdd0c463c4a7ab3aa7703c7 a8eb08c7-10a1-11ec-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=146860 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0