20.500.12556/DKUM-82073 Detekcija celulaz iz prebavil žuželk in njihov potencial za razgradnjo celuloze Detection of cellulases from insects gut and their potential for cellulose degradation V prebavilih večine žuželk so v naravi prisotne bakterije, ki pomagajo pri encimatski razgradnji biomase. Podrobnejše poznavanje encimov in možnosti heterolognega izražanja le-teh predstavlja velik potencial v biotehnologiji, kjer bi izražene encime lahko uporabili pri razgradnji celuloznih odpadkov. V okviru magistrske naloge smo želeli ugotoviti prisotnost genov za celulaze mikrobnega izvora v nabranem biološkem materialu (kobilice vrst Tettigonia viridissima, Decticus verrucivorus in Schistocerca sp. ter sviloprejke Bombyx mori) in jih klonirati v vektor, ki bi služil kot podlaga za potencialno ekspresijo iskanih celulaz. Na podlagi literature smo identificirali bakterije, ki se potencialno nahajajo v prebavilih kobilic in sviloprejk. Oblikovali smo filogenetsko drevo glikozil hidrolaz, ki so prisotne v identificiranih bakterijah, in izbrali 13 potencialno prisotnih (9 za kobilice in 4 za sviloprejke). Izolirali smo prebavila osebkov žuželk in ekstrahirali celokupni DNA. Prisotnost genov, ki kodirajo za glikozil hidrolaze v celokupnem genomu prebavila, smo preverjali z reakcijo PCR. Prisotnost smo ugotovili za skupno 11 od 13 izbranih celulaz (9 za kobilice in 2 za sviloprejke). V sklopu te naloge smo uspešno pomnožili gene, ki kodirajo za omenjene celulaze, modificirali vektor pET-22b(+) za ekspresijo celulaz v bakteriji Escherichia coli in tako doprinesli k razvoju sistema za ekspresijo encimov iz mikrobioma, ki se nahaja v prebavilih žuželk. In the gut of most insects, bacteria are naturally present and help to degrade ingested cellulose biomass. A more detailed knowledge of these enzymes and possibilities of their heterologous expression represents a great potential in biotechnology, wher the expressed enzymes could be used in decomposition of cellulose waste. The objective of this master thesis was to check presence of genes for microbial cellulases in collected material (grasshoppers Tettigonia viridissima, Decticus verrucivorus, Schistocerca sp. and silk worms Bombyx mori) and clone these genes in vector for potential expression of cellulases. We identified potential bacteria in insect's guts from literature. We designed phylogenetic tree of glycosyl hydrolases that are harboured by bacteria in insect's guts and chose 13 potentially present hydrolases (9 for grasshoppers and 4 for silk worms). We isolated insect's guts and extracted total DNA. We checked presence of genes with PCR reaction and confirmed 11 out of 13 present sequences (9 for grasshoppers and 2 for silk worms). Altogether, in this master thesis, we successfully amplified sequences coding for cellulases, constructed a modified version of the vector pET-22b(+) for expression of the glycosyl hydrolase(s) in Escherichia coli and thus contributed to the development of an expression system for enzymes, found in insects gut. biotehnologija celulaza celuloza mikrobiota prebavilo žuželk biotechnology cellulase cellulose microbiota insects gut true true false [A. Vaupotič] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-07-08 18:59:08 2022-08-31 13:47:28 2022-09-01 03:06:47 0000-00-00 00:00:00 2022 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 60:577.15: 595.71/.79(043.2) 119702531 MAG_Vaupotic_Ana_2022.pdf MAG_Vaupotic_Ana_2022.pdf 1 224F9B6A1D9BCBC167DD0D439B0EB712 6304e25b682074934c5eb1a6d5bfcb28cd764da22168a2a022a1aabea18594e6 9efcf39a-fee0-11ec-83e8-00155d000105 https://dk.um.si/Dokument.php?lang=slv&id=159266 Fakulteta za naravoslovje in matematiko 0 0 0