20.500.12556/RUL-137166 In vivo enkapsulacija specifičnih fragmentov DNA v bakterijski mikrorazdelek In vivo encapsulation of specific DNA fragments in bacterial microcompartment Bakterijski mikrorazdelki so primitivni proteinski organeli, ki jih sintetizira okrog 20 % bakterijskih vrst, katerih zaporedje genoma je določeno. Njihova glavna naloga je izboljševanje učinkovitosti posameznih metabolnih reakcij ali preprečitev akumulacije toksičnih metabolitov v citoplazmi. V osnovi jih delimo na tiste, ki sodelujejo v anabolizmu in na tiste, ki sodelujejo v katabolizmu celice. Med slednjimi je najbolj znan mikrorazdelek Pdu, ki je bil zaradi modularnosti uporabljen že za več biotehnoloških aplikacij. V magistrskem delu smo prikazali novo aplikacijo, in sicer enkapsulacijo specifičnega fragmenta DNA, kar bi lahko služilo kot osnova metode za odkrivanje interakcij protein-DNA ali kot ogrodje za metabolni inženiring. Za lažjo izolacijo smo podenoti mikrorazdelka PduA dodali peptidno značko Twin-Strep-tag, ki omogoča afinitetno izolacijo intaktnih mikrorazdelkov. Pokazali smo, da se fuzijski protein PduD(1-18)-LacI-EGFP učinkovito enkapsulira v mikrorazdelke in vivo ter po afinitetni izolaciji mikrorazdelkov ostane funkcionalen. Pripravili smo sistem dveh plazmidov v Escherichia coli, ki je aktiviran z dodatkom arabinoze v gojišče. Z enega plazmida se začnejo izražati geni za kvasno restriktazo I-SceI, protein Gam, podenote mikrorazdelka Pdu in gen za fuzijski protein PduD(1-18)-LacI-EGFP. Na drugem plazmidu sta eno ali dve restrikcijski mesti za kvasno restriktazo I-SceI, ki obdajata vezavna mesta za LacI. Z visokozmogljivim sekvenciranjem smo dokazali, da je DNA fragment z vezavnimi mesti za protein LacI v afinitetno izoliranih mikrorazdelkih Pdu obogaten glede na druge regije genoma, kar kaže na to, da je bil ta fragment uspešno enkapsuliran. Dokažemo tudi, da lahko v mikrorazdelek Pdu enkapsuliramo DNA z vezavnimi mesti za vsaj dva transkripcijska faktorja. Bacterial microcompartments are primitive proteinaceous organelles synthesized by approximately 20 % of bacterial species whose genomes have been sequenced. Their main function is to improve the efficiency of metabolic reactions or to accumulate toxic metabolic byproducts. In general, depending on their function, they are divided into those that perform anabolic or catabolic reactions. Among the latter, the Pdu microcompartment is the most studied, as it is used in many biotechnological applications due to its modularity. In this master's thesis, we demonstrate a new application, more specifically, the encapsulation of a specific DNA fragment in the microcompartment that could be used to study protein-DNA interactions or as a scaffold for metabolic engineering. To facilitate isolation, we tagged the major subunit of the microcompartment PduA with the peptide tag Twin-Strep-tag, which allows affinity isolation of intact microcompartments. We show that the fusion protein PduD(1-18)-LacI-EGFP is efficiently encapsulated in microcompartments in vivo and remains functional after affinity isolation of the microcompartments. We prepared a system of two plasmids in Escherichia coli. The first plasmid carries selected components under an arabinose-inducible promoter, genes for: Yeast nuclease I-SceI, phage protein Gam, subunits of the Pdu microcompartment and the fusion protein PduD(1-18)-LacI-EGFP. On the second plasmid, there are one or two restriction sites for the yeast endonuclease I-SceI flanking the binding sites for LacI. Using high-throughput sequencing, we demonstrate that in the affinity-isolated microcompartments, the fragment carrying LacI binding sites is enriched relative to other regions of the genome, suggesting that this fragment was truly encapsulated. In addition, we show that we can encapsulate a DNA fragment carrying at least two different transcription factors in the microcompartments. bakterijski mikrorazdelek enkapsulacija DNA transkripcijski faktor bacterial microcompartment DNA encapsulation transcription factor true false false [J. Otoničar] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2022-06-04 07:15:06 2022-06-04 07:15:25 2022-09-15 04:14:44 0000-00-00 00:00:00 2022 0 Ljubljana 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 601.4:577.21:582.23(043.2) 203522 110798083 23007.pdf 23007.pdf 1 134AEB23C9900FB2A327EDF31A424901 4d72d0d0a4a97c1efcbcdc558587b52168eb81e91b791dd90d4970909e537f1d 3ef3f544-e3c5-11ec-8aca-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=157030 Biotehniška fakulteta 0 0 0