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[{"key": "dc.contributor.advisor", "value": "Penttinen, Reetta", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Ruotsalainen, Pilvi", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Jalasvuori, Matti", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Eklund, Tiia", "language": "", "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Elomaa, Hanna", "language": "", "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2018-06-08T10:11:55Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2018-06-08T10:11:55Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2018", "language": "", "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/58460", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Bakteeri-infektiot hoidetaan nyky\u00e4\u00e4n p\u00e4\u00e4asiassa antibiooteilla. Antibioottien k\u00e4yt\u00f6n aiheuttama valintapaine on johtanut antibioottiresistenttien bakteereiden runsastumiseen, mik\u00e4 hankaloittaa infektioiden hoitoa. Antibioottiresistenssi onkin maailmanlaajuinen kasvava ongelma. \u03b2-laktamaasit ovat entsyymej\u00e4, jotka hajottavat \u03b2-laktaamirenkaan est\u00e4en n\u00e4in \u03b2-laktaami-antibiootin toiminnan. \u03b2-laktaami-antibiootit, kuten penisilliinit ja kefalosporiinit, ovat yleisesti k\u00e4ytettyj\u00e4 antibiootteja, joiden k\u00e4ytt\u00f6 on aiheuttanut \u03b2-laktamaaseja tuottavien laajakirjoisten \u03b2-laktamaasibakteerien (ESBL, engl. extended spectrum \u03b2-lactamase) yleistymist\u00e4. ESBL-bakteerit tuottavat \u03b2-laktamaaseja, jotka antavat laajakirjoisen resistenssin useita \u03b2-laktaameja vastaan. CRISPR-Cas9-systeemi on alun perin bakteerien luonnollinen puolustusmekanismi vierasta DNA:ta vastaan. CRISPR-Cas9-systeemi\u00e4 hy\u00f6dynnet\u00e4\u00e4n geenien katkaisemisessa targetoimalla kohdegeeni sille komplementaarisen crRNA-sekvenssin (engl. CRISPR RNA) avulla. Kohdegeeni katkaistaan Cas9-entsyymin avulla, jolloin geenin transkriptio estyy. T\u00e4ss\u00e4 ty\u00f6ss\u00e4 oli tavoitteena katkaista pEC3- ja pEC15-plasmideissa olevat blaTEM-1C- ja blaTEM-52B-antibioottiresistenssigeenit CRISPR-Cas9-systeemill\u00e4. Kohdegeenit tuottavat ampisilliinia hajottavia TEM-tyypin \u03b2-laktamaaseja ja niiden sekvenssit eroavat toisistaan muutaman aminohapon osalta. Tavoitteena oli tutkia, kuinka hyvin TEM-entsyymien konservoitunutta kohtaa targetoivaksi suunniteltu crRNA-sekvenssi toimii. E. coli BL21 Gold (pEC3)- ja BL21 Gold (pEC15)-bakteerikantoihin transformoitiin CRISPR-Cas9-systeemin sis\u00e4lt\u00e4v\u00e4 crRNA-plasmidi. Systeemin toimiessa Cas9-entsyymi pilkkoi resistenssigeenin. Kontrollisoluihin transformoitiin kontrolliplasmidi, josta puuttui crRNA-sekvenssi. Systeemin tehokkuutta arvioitiin vertaamalla crRNA:n sis\u00e4lt\u00e4vien solujen ja kontrollisolujen bakteerim\u00e4\u00e4ri\u00e4 antibioottialtistuksen j\u00e4lkeen. CRISPR-Cas9-systeemin crRNA:lla kohdennettiin hyvin molempia blaTEM-1C- ja blaTEM-52B-resistenssigeenej\u00e4. Solut menettiv\u00e4t ampisilliiniresistenssin ja kuolivat ampisilliiniselektiomaljalla. blaTEM-52B-resistenssigeenin katkaisemisessa saavutettiin hieman parempi tulos kuin blaTEM-1C-resistenssigeenin katkaisemisessa, sill\u00e4 pEC3-linjan solum\u00e4\u00e4r\u00e4 laski 102 cfu/ml ja pEC15-linjan 103 cfu/ml. Pes\u00e4ke-PCR:ll\u00e4 ja agaroosigeelielektroforeesilla (AGE, engl. agarose gel electrophoresis) tarkistettiin, oliko crRNA pysynyt eloonj\u00e4\u00e4neiss\u00e4 transformoiduissa soluissa. Tuloksista huomattiin, ett\u00e4 suurimmasta osasta bakteereista crRNA oli kadonnut mahdollisesti jonkun mutaation seurauksena. N\u00e4ytteiss\u00e4, joissa crRNA oli tallella, CRISPR-Cas9-systeemi ei ollut jostain muusta syyst\u00e4 kuin puuttuvan crRNA:n vuoksi toiminut. Viel\u00e4 tarvitaan lis\u00e4tutkimusta, jotta voidaan selvitt\u00e4\u00e4 mink\u00e4 vuoksi k\u00e4ytetyn crRNA:n avulla ei saatu poistettua ampisilliiniresistenttiytt\u00e4 kokonaan bakteerikasvustoissa.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Antibiotics are nowadays the main treatment against bacterial infections. Antibiotic resistance is an increasing problem and it complicates the treatment of infections. Especially \u03b2-lactams, such as penicillins and cephalosporins, are commonly consumed antibiotics and their use has enhanced the spreading of ESBL-bacteria. Those bacteria have resistance against many \u03b2-lactam antibiotics. The reason is that they produce \u03b2-lactamase enzymes which can degrade various \u03b2-lactams. CRISPR-Cas9 system is a natural bacterial defense mechanism against foreign DNA. The system can be used for genetic modification. The CRISPR RNA (crRNA) of CRISPR-Cas9 system binds complementary to the target gene. The target gene is then cut by Cas9 enzyme, resulting in the interruption of the transcription of the gene. The aim of this experiment was to degrade ampicillin resistance genes blaTEM-1C (pEC3) and blaTEM-52B (pEC15) by using a targeted CRISPR-Cas9 system. The pEC3 and pEC15 plasmids were in E. coli BL21 Gold host bacteria. These host bacteria were transformed with a crRNA-plasmid expressing CRISPR-Cas9 system. Transformed bacteria were cultured in ampicillin selection plate. When the system was functional, the expression of resistance genes was stopped and the bacteria died in the ampicillin selection. The control bacteria were transformed with a control plasmid without crRNA. By comparing the amount of viable bacteria transformed with crRNA or control plasmid, the efficiency of CRISPR-Cas9 system was estimated. According to our results, the bacteria quantity of BL21 Gold (pEC3) dropped off 102 cfu/ml and BL21 Gold (pEC15) 103 cfu/ml. These results indicate that the crRNA of the CRISPR-Cas9 system cut efficiently both targeted resistance genes, but slightly better the blaTEM-52B gene. These cells lost the resistance to ampicillin and did not survive in ampicillin selection. Viable cells were analyzed by using colony PCR and agarose gel electrophoresis (AGE) to see if crRNA was still present in these cells. The results showed that crRNA was lost from the majority of samples probably as a consequence of mutation. Some of the transformed samples still contained crRNA, which indicate that the CRISPR-Cas9 system did not work in these cells for some other reason than missing crRNA. It is necessary to find out why CRISPR-Cas9 system with crRNA used in this experiment did not target all the bacteria carrying pEC3 and pEC15 plasmids.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by Paivi Vuorio (paelvuor@jyu.fi) on 2018-06-08T10:11:55Z\nNo. of bitstreams: 0", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2018-06-08T10:11:55Z (GMT). No. of bitstreams: 0\n Previous issue date: 2018", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "29", "language": "", "element": "format", "qualifier": "extent", "schema": "dc"}, {"key": "dc.language.iso", "value": "fin", "language": null, "element": "language", "qualifier": "iso", "schema": "dc"}, {"key": "dc.rights", "value": "In Copyright", "language": "en", "element": "rights", "qualifier": null, "schema": "dc"}, {"key": "dc.subject.other", "value": "ampisilliiniresistenssi", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "ESBL", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "CRISPR-Cas9", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "blaTEM-1C", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "blaTEM-52B", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "\u03b2-laktamaasi", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "blaTEM1-C- ja blaTEM-52-B-antibioottiresistenssigeenien sammuttaminen CRISPR-Cas9-systeemill\u00e4", "language": "", "element": "title", "qualifier": null, "schema": "dc"}, {"key": "dc.type", "value": "bachelor thesis", "language": null, "element": "type", "qualifier": null, "schema": "dc"}, {"key": "dc.identifier.urn", "value": "URN:NBN:fi:jyu-201806083115", "language": "", "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Bachelor's thesis", "language": "en", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Kandidaatinty\u00f6", "language": "fi", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Matemaattis-luonnontieteellinen tiedekunta", "language": "fi", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Faculty of Sciences", "language": "en", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Bio- ja ymp\u00e4rist\u00f6tieteiden laitos", "language": "fi", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Department of Biological and Environmental Science", "language": "en", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "University of Jyv\u00e4skyl\u00e4", "language": "en", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Solu- ja molekyylibiologia", "language": "fi", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Cell and molecular biology", "language": "en", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "yvv.contractresearch.funding", "value": "0", "language": "", "element": "contractresearch", "qualifier": "funding", "schema": "yvv"}, {"key": "dc.type.coar", "value": "http://purl.org/coar/resource_type/c_7a1f", "language": null, "element": "type", "qualifier": "coar", "schema": "dc"}, {"key": "dc.rights.accesslevel", "value": "openAccess", "language": null, "element": "rights", "qualifier": "accesslevel", "schema": "dc"}, {"key": "dc.type.publication", "value": "bachelorThesis", "language": null, "element": "type", "qualifier": "publication", "schema": "dc"}, {"key": "dc.subject.oppiainekoodi", "value": "4013", "language": "", "element": "subject", "qualifier": "oppiainekoodi", "schema": "dc"}, {"key": "dc.subject.yso", "value": "geenitekniikka", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "bakteerit", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "geenit", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "resistenssi", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "l\u00e4\u00e4keresistenssi", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.rights.url", "value": "https://rightsstatements.org/page/InC/1.0/", "language": null, "element": "rights", "qualifier": "url", "schema": "dc"}]
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