Browsing by master's degree program "Mikrobiologian ja mikrobibiotekniikan maisteriohjelma"

Microbiology is included in the subject of biology in lower secondary school as part of the National Core Curriculum for Basic Education (POPS). Microbiology teaching has traditionally relied on inquiry-based methods and practical work, where inquiry is highlighted in POPS. In this study, a survey of teachers investigated microbiology teaching and inquiry-based methods in lower secondary school. The study focused on these areas: 1. What groups of microbes are mentioned and in what biological contexts? 2. What methods are used in teaching microbiology? 3. How much are inquiry-based methods used and does the amount vary regionally? 4. What type of inquiry-based teaching is used? and 5. What are the potential limitations in delivering inquiry-based teaching? The data were collected by an online survey for biology teachers in lower secondary schools around Finland and 36 responses were received. The data were analyzed mainly by qualitative content analysis which was supported by quantitative and regional analysis. According to the results, the coverage of microbial diversity is wide in biology teaching but Archea may receive less attention. Microbiology is taught well in many biological contexts but least in the context of evolution and development of life. The teaching methods are diverse, and many different practical activities are carried out. Inquiry-based methods were utilized by 97.2% of teachers and the amount does not vary regionally. Structured inquiry is used the most in microbiology teaching and the majority of the teachers found inquiry-based methods valuable. The amount of inquiry is limited by a lack of time, size and heterogeneity of the student groups, lack of equipment and workspace. The results indicated that the teaching of microbiology in lower secondary school is diverse and inquiry-based methods are common, but limitations were expressed. Solutions in response to these concerns could be, for instance, virtual activities, improved learning materials and more collaboration between universities and schools which is now poor. There are so far no other published studies about this in Finland. In the future, it would be interesting to study further how inquiry-based methods and the teacher’s self-efficacy and own training in these areas affect the outcomes of microbiology education. Such research could then be used to improve teacher education to address the limitations presented in this thesis.

The spread of antibiotic resistance in bacteria is a global problem. Horizontal gene transfer (HGT) is the main mechanism implicated in the spread of antibiotic resistance genes (ARG). This study is related to a doctoral thesis project that studies HGT in wastewater microbial community by conducting a microcosm experiment that uses Emulsion, Paired-Isolation and Concatenation PCR (epicPCR) to monitor the spread of ARGs between species. The aim of this study was to introduce synthetic epicPCR primer binding sites inside various ARGs and to test the function of the encoded proteins. The goal was to maintain sufficient protein function, i.e., antibiotic resistance despite the modifications, which allows the further use of modified ARGs in microcosm experiment. The ARGs selected for modifications were dfrB2, ermB, ermC, sul1 and sul2. Sequence-based prediction method was applied to find regions that tolerate insertions inside the proteins encoded by ARGs. The modified ARGs carried in plasmid pUC19 were introduced to Escherichia coli DH5α, which was used as the host in antibiotic susceptibility testing. Antimicrobial gradient method was used to test the antibiotic susceptibility of the strains and to verify the function of the proteins. Six ARGs modified in this study encoded for functional proteins that conferred antibiotic resistance while three modified ARGs did not. Two out of four proteins with insertions in predicted permissive stretches in the middle of a protein maintained their function. The six functional, antibiotic resistance conferring genes designed in this study can be used in further studies utilizing epicPCR. Based on the results of this study, sequence-based prediction method for finding permissive stretches seems useful, but it does not guarantee that the protein function is maintained.

Biologia on tieteenalana jatkuvassa muutoksessa. Geenitutkimusten kehittyessä saadaan uutta tietoa eliöiden perimästä ja sukulaissuhteista, jolloin niitä voidaan myös luokitella tarkemmin. Opinnäytetyössä tarkastellaan biologian tieteenalalla tapahtuvien muutoksien vaikutuksia biologiaan oppiaineena peruskoulussa ja lukiossa. Tutkimuksessa selvitän, miten biologian opettajat suhteutuvat päivitettyyn eliöiden luokitteluun ja se otettu osaksi opetusta. Lisäksi tavoitteenani on selvittää mitkä eliöryhmät opettajat kokevat merkittäväksi opettaa kullakin opetusasteella, ja ovatko jotkin eliöryhmät opettajien mielestä merkityksettömiä yleissivistävän koulutuksen kannalta. Selvitän myös mitä aineistoja biologian aineenopettajat käyttävät merkittävimpänä lähteenään opetuksessa. Tutkimus toteutettiin puolistrukturoituna kyselylomakkeena, jossa oli sekä avoimia kysymyksiä, että monivalintakysymyksiä. Tutkimuksen taustatiedot osio on tehty kirjallisuuskatsauksena luokittelun muutokseen liittyviin artikkeleihin sekä peruskoulun ja lukion opetussuunnitelmaan pohjautuen. Tutkimuksen tuloksia analysoitiin sekä kvantitatiivisesti että kvalitatiivisesti. Osa opettajista ei ole ollut tietoinen eliökunnan luokittelun päivityksestä, mikä on vaikuttanut sen käyttöönottamiseen opetuksessa. Päivitetty luokittelu on koettu haastavimmaksi yläkoulun opettajien keskuudessa, eikä sitä ole otettu yhtä laajasti käyttöön kuin lukioissa. Opettajilla on melko yhtenäinen käsitys siitä, mitä eliöryhmiä tulisi opettaa kullakin opetusasteella. Vastausten perusteella opettajat käyttävät tiedonlähteenä pääasiassa opettajille tuotettuja materiaaleja. Tämän vuoksi olisi tärkeää, että keskeisistä uudistuksista tiedotetaan riittävästi ja niistä tehtäisiin helposti saatavilla olevia lisämateriaaleja.

This project focuses on development of novel split intein systems for selection of biological activities utilizing protein splicing. Protein splicing is phenomenon that occurs naturally inside the cell and the reaction is catalyzed by inteins, which connect C-terminal and N-terminal exteins with a peptide bond. The activity of the interrupted protein, consisting the exteins, can be restored after the intein is excised and the peptide bond links the exteins together. This occurrence can be used for selection of cells based on different activities including antibiotic resistance. The project aims to insert an intein in antibiotic resistance gene which could allow controlling the protein activity of the antibiotic resistance gene by protein splitting. This method is based on inserting an intein to the antibiotic resistance conferring enzyme which makes the protein inactive. Creating two separate plasmids that include the intein sequence can be transformed into bacterial cells. Other plasmid includes a deletion in the intein sequence and the cells that include this plasmid only, are not able to survive in the presence of an antibiotic. This is due to inactivity of the intein and thus the inactivity of the enzyme that confers the resistance. Incorporating a second plasmid that includes the corresponding sequence to the deletion, the intein activity can be recovered and thus the protein activity. By this method with cotransformation, both plasmids are transformed simultaneously which recovers the intein activity and further the antibiotic resistance. This could be used for the cell selection since only the cells that harbor both of the two complementary plasmids could restore the antibiotic resistance.

Filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a crucial production organism for enzymes used in industrial applications, such as in feed, food, textile, and biofuel production, due to its ability to secrete high amounts of homologous and heterologous enzymes. Therefore, development of genetic tools to improve the properties of industrial T. reesei strains for even better production yields is essential. In this study, a polyethylene glycol mediated CRISPR-Cas9 transformation method for industrial T. reesei production strains was aimed to be optimised by testing an alternative Cas9 enzyme and varying the stoichiometry and total amount of Cas9 enzyme and single guide RNA in the ribonucleoprotein complex. Correct integration of the gene constructions in the obtained transformants was determined by colony PCR and Southern blot analysis. In addition, two selected background activity encoding genes, endoglucanase 6 and α-glucuronidase 1, were individually deleted from T. reesei xylanase production strain utilising the improved CRISPR-Cas9 transformation protocol. The effect of background activity deletions on the strain growth and protein production were analysed from culture supernatants by pH measurement, Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and enzyme activity assays. An improved CRISPR-Cas9 transformation protocol for T. reesei was successfully established basing on high number of transformants and improved DNA integration fidelity. No negative effects were observed in the growth or protein production properties of the background activity deletion strains compared to the xylanase production strain. Thus, further cleansing of T. reesei secretome can be continued to refine the industrial production strains.

The increasing use of antimicrobials causes a heavy pollution load on the environment and can enhance antimicrobial resistance of pathogenic bacteria, thus having a negative impact on human and animal health. Antibiotic concentrations in the environment are constantly monitored, however traditional chemical analyses fail to provide data on the bioavailability of antimicrobials. Whole-cell bacterial bioreporters have been developed to detect a wide variety of environmental pollutants including antimicrobials. These living, genetically engineered organisms can also be used for the measurement of the bioavailable fraction in a sample and thus bioreporters could give insights on the role of antimicrobial pollution in the dissemination of antimicrobial resistance. The aim of this study was to design and construct an improved bioluminescent bioreporter for detection of macrolide antibiotics. The mrx gene and the mph(A)R repressor gene were coupled with the mph(A) promotor of the macrolide resistance operon mph(A) and the reporter genes of the luciferase operon luxCDABE. The main objective was to determine whether Mrx, the hydrophobic and putative transmembrane transport protein of the macrolide resistance operon mph(A), would improve the sensitivity and reduce the induction time. Another aim was to optimize the use of three existing bioreporters with other macrolides than erythromycin, which was used earlier in testing their performance. The mrx-mph(A)R fragment was cloned into the pmph(A)luxCDABE vector, and the bioreporter plasmid was introduced to Escherichia coli strain DH10B. After verification of the construct pmph(A)luxCDABE-mrx-mph(A)R, the usability of the new whole-cell biosensor was compared against the three existing macrolide bioreporters with three different macrolide and lincosamide antibiotics, erythromycin, tylosin and clindamycin. The cloning of the new bioluminescent bioreporter for macrolides was performed successfully. However, the addition of erythromycin, tylosin or clindamycin to a suspension of E. coli DH10B(pmph(A)luxCDABE-mrx-mph(A)R) did not stimulate the expression of the lux genes. High concentrations of all three antibiotics triggered a light response with the existing bioreporters although the response was slow. The results indicated that further studies on the mrx gene and its encoded Mrx protein are still needed. The response of the mph(A) operon to other macrolide and lincosamide antibiotics than erythromycin was a positive and encouraging finding, since this enables detection of other synthetic macrolides than erythromycin as well as lincosamides with the existing bioreporters after optimization.

Kaupunkien ja maatalouden lähteistä johtuva vesistöjen mikrobiologinen kontaminaatio muodostaa merkittävän riskin ihmisten, eläinten ja ympäristön terveydelle. Saastumisen lähteiden tunnistaminen auttaa ymmärtämään sen luonnetta ja auttaa estämään uusia saastumisia. Mikrobilähteiden jäljitys tunnistaa tehokkaasti vesistöjen saastumisen lähteet geenimerkkien kautta, tarjoten tarkan ja herkän tavan tunnistaa mikrobiperäisen saastumisen luonnetta. Tässä tutkimuksessa valittiin 10 geenimerkkiä, jotka kohdistuivat viiteen taudinaiheuttajaan ja viiteen mikrobisaasteeseen vesiympäristöissä. Kun valittujen geenimerkkien alukkeiden ominaisuudet eivät olleet yhteneväisiä SmartChip qPCR-asetusten kanssa, suunniteltiin uudet alukkeet NCBI:n Primer BLAST -työkalulla valitun geenimerkin havaitsemiseksi. Sopivilla ominaisuuksilla varustetut alukkeet analysoitiin in silico analyysillä käyttäen primer-BLASTN työkalua, jotta alukkeiden tarkkuus kohde geeniin varmistettiin. qPCR tuotteet analysoitiin Sanger-sekvensoinnilla, jotta voitiin havaita, olivatko odotettuja. Tämä analyysi tarjosi tietoa alukkeiden mahdollisesta väärin sitoutumisesta. Tämä kymmenen alukkeen kokonaisuus suunniteltiin korkean yhteensopivuuden saavuttamiseksi SmartChip qPCR:n sykliolosuhteiden kanssa. Se luotiin myös tarkasti heijastamaan geenejä ja organismeja, jotka esiintyvät ympäristön vesinäytteissä.

Rift Valley fever virus (RVFV) is a mosquito-borne virus of the order Bunyavirales with a tripartite (-)ssRNA genome. It infects humans and cattle, causing a febrile disease with symptoms ranging from mild to severe. Safe and efficient human vaccines have not yet been developed, which underlines the importance of gaining a clear understanding of the viral antigenic surface. One significant challenge for RVFV research is posed by the costly and time-consuming biosafety precautions warranted by the pathogenicity of the virus. The surface of RVFV, formed by the two viral envelope glycoproteins Gn and Gc, could also be studied with a non-pathogenic model, such as a virus-like particle (VLP). A VLP is a macromolecular complex that resembles the virus, especially with respect to its outer structure, but lacks the viral genome. In this work, RVFV VLPs were produced by transient transfection of mammalian cells with genes encoding RVFV glycoproteins Gn and Gc. The objective was to design an optimized production and purification pipeline for RVFV VLPs to elucidate their structure by cryo-electron microscopy. To optimize the VLP production and purification, the effect of sample harvest times and DNA-to-cell ratios of transfection on RVFV glycoprotein expression was examined. Several methods were tested for VLP sample concentration and purification. VLPs were successfully detected in the purified samples by immuno-electron microscopy. Despite some challenges related to sample purity and scarcity of VLPs in samples, which prevented analyses by cryo-electron microscopy, the expression system described in this thesis has great potential to streamline RVFV VLP sample preparation for electron microscopy and to accelerate vital research into the structural properties of this emerging pathogen.

Yersinia pseudotuberculosis voi aiheuttaa ihmiselle suolistoinfektion, yersinioosin. Se tarttuu kontaminoituneiden elintarvikkeiden välityksellä, ja Suomessa on todettu useita epidemioita viimeisen 20 vuoden aikana. Yersinia-suku kuuluu Enterobacteriaceae-heimoon, ja siihen kuuluu tällä hetkellä 18 lajia. Y. pseudotuberculosis-lajin lisäksi sukuun kuuluu kaksi ihmispatogeenia, Yersinia enterocolitica ja Yersinia pestis. Y. enterocolitica on myös elintarvikevälitteinen patogeeni, Y. pestis puolestaan tunnetaan ruton aiheuttajana. Y. pseudotuberculosis kasvaa hyvin jääkaappilämpötiloissa, jolloin nykyaikaiseen elintarvikehygieniaan oleellisena osana kuuluva kylmäsäilytys ei estä sen lisääntymistä elintarvikkeissa. Sen vuoksi onkin tärkeä tutkia tekijöitä, joilla on merkitystä Y. pseudotuberculosis-bakteerin kylmänsiedossa ja muissa stressiolosuhteissa. RNA-helikaasit ovat proteiineja, jotka avaavat kaksijuosteista RNA:ta. Niihin kuuluu useita proteiiniperheitä, mutta suurin niistä on DEAD-box-helikaasiperhe. DEAD-box-helikaaseja löytyy kaikista eliöryhmistä, ja ne osallistuvat kaikkiin RNA-metabolian vaiheisiin. Ne ovat ATP-riippuvaisia helikaaseja ja ne kykenevät avaamaan vain lyhyitä kaksijuosteisia alueita. Y. pseudotuberculosis IP32953- genomi sisältää viisi DEAD-box-helikaasia koodaavaa geeniä, csdA, dbpA, rhlB, rhlE ja srmB. RhlB osallistuu mRNA-molekyylien hajottamiseen osana RNA-degradosomia. Muut proteiinit osallistuvat pääasiassa ribosomin rakentumiseen. Escherichia coli -bakteerilla aiemmin tehdyn tutkimuksen perusteella tiedetään, että CsdA:n ja SrmB:n puuttuminen soluista saa aikaan kylmäherkän fenotyypin. CsdA:n roolia on tutkittu myös Y. pseudotuberculosis-lajilla, jossa toimimaton CsdA heikentää kasvua kylmässä. Tämän tutkielman tarkoituksena oli tutkia RNA-helikaasien vaikutusta Y. pseudotuberculosis IP32953-kannan kasvuun erilaisissa stressiolosuhteissa. RhlE-, DbpA- ja SrmB-deleetiomutanttikantoja kasvatettiin optimilämpötilan (28 °C) lisäksi kylmässä (3 °C), korkeassa suolapitoisuudessa sekä alhaisessa ja korkeassa pH:ssa. Kasvun aikana mitattiin bakteerien kasvua OD600nm-mittausten avulla, ja aineiston perusteella kannoille piirrettiin kasvukäyrät kaikissa olosuhteissa. Lisäksi aineistosta laskettiin käyrän alle jäävä pinta-ala, jonka perusteella tutkittiin kasvuerojen tilastollista merkitsevyyttä Studentin t-testillä. Kannoille määritettiin myös kasvunopeudet eri olosuhteissa. Deleetiomutanttien kasvussa huomattiin eroa alhaisessa lämpötilassa, jossa kaikki deleetiomutanttikannat tilastollisesti merkitsevästi kasvoivat villityypin kantaa huonommin. Lisäksi ΔsrmB -kanta kasvoi tilastollisesti merkitsevästi heikommin optimilämpötilassa ja korkeassa pH:ssa. Näyttääkin siis siltä, että DEAD-box-helikaasit ovat Y. pseudotuberculosis-bakteerilla suuremmassa roolissa kuin E. coli-bakteerilla, ja niitä tarvitaan erityisesti alhaisissa lämpötiloissa.

The ever-increasing spread of antibiotic resistant bacteria creates a constant demand for new sources for antimicrobial drugs. Phages are a natural source for antibacterial proteins, but also produce a variety of unknown compounds, referred to as “hypothetical proteins of unknown function” (HPUF). HPUFs usually consist of structural proteins, but also small polypeptides that inhibit bacterial growth during infection. These peptides could be utilized in the discovery of new antimicrobial molecules. However, the current methods used for the screening of such proteins are time consuming and unreliable, making this a fairly unpopular option to utilize. In this study, a new NGS (Next Generation Sequencing) based assay for the screening of phage derived bacteriotoxic proteins was developed and tested by performing two separate experiments together with a previously used plating assay as a comparative method. A preliminary experiment was performed as a proof of principle, with five known toxic and five non-toxic genes. After this, the methods were compared by screening 23 previously identified HPUF genes of phage fHy-Eco03. In the plating assay genes were screened individually by observing growth of bacterial transformants upon gene expression. In the NGS assay genes we screened simultaneously by transforming them to E. coli cells as a pooled sample. Results were obtained with bioinformatics. Toxic genes were expected to be identified through a decrease in sequence read amount, as a consequence of bacterial growth inhibition. In the pre-experiment a difference between toxic and non- toxic proteins was not observed. The results between the NGS and plating assay in the screening of phage fHy-Eco03 genes, were similar and resulted in the identification of one toxic protein. The inconsistent results are probably an outcome of lac promoter repression by glucose supplementation, thus only highly toxic genes show an inhibitory effect. Despite this the NGS assay outperformed the plating assay in both accuracy and efficiency. The NGS assay has high potential to be used as a screening assay for phage derived toxic genes, however further optimization and validation is required, by firstly selecting compatible media and secondly by re- testing with different phages and host bacteria.