Browsing by master's degree program "Master 's Programme in Microbiology and Microbial Biotechnology"

Perusopetuksen opetussuunnitelman perusteet (POPS) painottaa biologian opetuksen kohdalla tutkivaa oppimista ja tutustumista biologialle ominaisiin tutkimusmenetelmiin. POPS:n biologian sisältöalueisiin kuuluu myös mikrobiologiaa. Tässä tutkimuksessa selvitettiin sisällönanalyysin keinoin miten yläkoulun biologian Koodi- ja Elo-oppikirjasarjojen tehtävissä ilmeni mikrobiologia ja tutkimuksellisuus. Tutkimuskysymykset olivat: 1. Kuinka paljon mikrobiologiaan liittyviä tehtäviä on ja mihin aiheeseen ne liittyvät? 2. Kuinka paljon mikrobiologian tutkimuksellisia tehtäviä on ja millaista tutkimuksellisuutta ne edustavat? 3. Onko mikrobiologian tehtävien määrällä ja laadulla eroavaisuutta kirjasarjojen välillä? Tulosten perusteella pohdittiin vastasivatko oppikirjat POPS:n sisältöjä ja tavoitteita mikrobiologiaan liittyvien tehtävien osalta. Molemmissa kirjasarjoissa oli mikrobiologiaan liittyviä tehtäviä lähes yhtä paljon. Koodi-kirjasarjassa tehtäviä oli selvästi eniten Elämä-kirjassa ja Elo-kirjasarjassa tehtäviä oli eniten Ihminen-kirjassa. Koodi-kirjasarjassa erilaisia mikrobiryhmiä oli käsitelty hieman kattavammin ja tasaisemmin kuin Elo-kirjasarjassa. Elo-kirjasarjasta myös puuttuivat kokonaan yhteen POPS:n sisältöalueeseen, elämän kehitykseen ja evoluutioon, liittyvät mikrobiologiset tehtävät, joita Koodi-kirjasarjassa oli useita. Sen sijaan vain Elo-kirjasarjassa oli POPS:ssa mainittua biotekniikka-sisältöä mikrobiologiaan liittyen. Tutkimuksellisia tehtäviä oli molemmissa kirjasarjoissa vähän alle puolet mikrobiologian tehtävistä. Elo-sarjassa niitä oli hieman enemmän, tutkimuksellisuus oli monipuolisempaa ja myös kokonaisia tutkimuksia oli useita, toisin kuin Koodi-kirjasarjassa. Molemmissa kirjasarjoissa oli useita mikroskopointitehtäviä. Tuloksista voitiin päätellä, että molemmat kirjasarjat vastaavat POPS:n tutkimuksellisuustavoitteeseen, vaikkakin Elo-kirjasarja hieman paremmin. Mikrobiologian aihesisältöjen osalta kirjoissa oli painotuseroja ja joitain puutteita verrattuna POPS:n sisältöihin. Tutkimuksen tulokset voivat olla biologian opettajan apuna oppikirjan valinnassa ja sen POPS:n vastaavuuden tarkistamisessa. Jatkotutkimuksena oppikirjojen tekstin analysointi antaisi kokonaiskuvan oppikirjojen mikrobiologian sisällöstä. Oppikirjojen mikrobiologian sisältöjen näkymistä opetuksessa voisi myös tutkia.

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.

Methanococcus maripaludis is a hydrogenotrophic methanogen which has become a model organism for archaea and for the study of methanogenesis. New genetic and genomic tools hold promise to convert M. maripaludis into a host organism for synthetic biology and metabolic engineering. However, an extensive characterization of suitable promoters in this organism for their use in metabolic engineering has been lacking. In this study, the strength of 25 promoter sequences was quantified by use of a β-glucuronidase reporter gene assay, establishing it as an adequate method for this purpose. PglnA seemed to evade the control exerted by the native transcriptional regulator NrpR and thus became the strongest promoter of all tested. Pmtr, Pmcr, , Pmcr_JJ and Ppor_JJ could also be regarded as strong promoters in this organism. On the other hand, inclusion of a putative transcription factor downstream of the eha operon increased the strength of Peha and Peha_JJ, suggesting its role as the activator of the operon. Overall, these results provide valuable information for the implementation of genetic, metabolic and promoter engineering in M. maripaludis. Genetic manipulation of M. maripaludis was done via a recently developed CRISPR/Cas toolbox, serving as an example of its efficiency.

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.

Water moulds belonging to genus Saprolegnia are known to be opportunistic fish pathogens and cause a deadly disease called saprolegniosis. Saprolegniosis is found in both wild and farmed fish as fish farms use natural waters as their fish-farming waters. Majority of cases of saprolegniosis found in fish farms are caused by Saprolegnia parasitica which is currently identified from dead fish by microscopic methods. This master's thesis was done for Finnish Food Authority. The aim of this thesis was to compare quantitative PCR (qPCR) methods in detection of S. parasitica and to evaluate the applicability of the best method. qPCR allows for better species identification than the microscopic method and it also enables the quantification of water mould DNA. The study was performed for three qPCR methods amplifying the gene region encoding S. parasitica aquatic mould 28S rRNA. Of the three methods, the best was selected based on specificity and sensitivity tests. The best method was used to detect and quantify S. parasitica in water samples from four Finnish fish farms. By this, the suitability of the method for water samples was confirmed. Also, data about the occurrence of S. parasitica water mould on fish farms was also collected. Based on the studies performed, the Roc-qPCR method based on the method developed by Rocchi et al. (2017) was selected as the best. The Roc-method was highly specific in identification of S. parasitica. With Roc- method, very low concentrations of S. parasitica DNA was quantified from fish farm water samples within good confidence. DNA amount of S. parasitica was statistically significantly higher in the effluent waters of fish farms, in which S. parasitica DNA levels were highest when water mould was detected in the fish farm fish. These results confirmed the notion that S. parasitica increases in fish. This increase of spores could be reliably detected by the Roc method. In the study, water temperature was not found to correlate with S. parasitica DNA, which may be due to the lack of data in the study. On the other hand, the occurrence of water mould is influenced by several other factors in addition to temperature, which were not considered in this study. Based on the results of this study, the Roc-qPCR method is a specific and sensitive method for the detection of S. parasitica from water samples. Roc-method is a potential method for use when designing of saprolegniosis control measures in fish farms. Further research is needed about the factors associated with the presence of S. parasitica in fish farms.

The gut microbiota is important for human health, participating in many important functions, such as digestion, and is strongly modulated by the diet. The consumption of red and processed meat should be reduced due to both health and environmental reasons. Red meat could be partially replaced with legumes, as they are rich in protein. In addition, legumes are a good source of fibre and increasing their consumption would increase fibre intake. Here we aimed to study the effects of a partial replacement of red and processed meat with legumes on the gut microbiota composition in Finnish healthy men. The study was a 6-week randomized clinical trial in parallel design and included two groups with diet supplemented either with red and processed meat (760 g/wk) or a lower amount of red and processed meat (200 g/wk) and legume products containing the corresponding amount protein as in 560 grams of red and processed meat. Both diets provided 25% of the participants’ daily protein intake. The microbiota composition was analysed before and at the end of the intervention period from faecal samples. In total 102 participants finished the study. The group with the diet containing legume products showed a significant reduction in alpha diversity (p=0.029) and in the relative abundance of the genus Prevotella (false discovery rate (FDR)-corrected p-value (p-FDR) =0.130) and Ruminococcaceae NK4A214 group (p-FDR=0.130) when comparing before and after the intervention period. No significant changes were seen in the meat-based diet group. When comparing the two diet groups at the end of the intervention period we observed a significantly higher relative abundance of the genus Agathobacter (p-FDR=0.023), Coprococcus ( p-FDR=0.154) and Ruminiclostridium (p-FDR=0.154) in the meat-based diet group, while the genus Bacteroides (p-FDR=0.112) and Ruminococcaceae UCG.013 group (pFDR=0.066) showed higher relative abundance in the legume-based diet group. In conclusion, our results show that even a partial replacement of red and processed meat affects the composition of the gut microbiota.

Single nucleotide polymorphism (SNP) is DNA variation of a single nucleotide. SNPs are the most common mutations and millions of single nucleotide differences distinguish humans genetically from each other. Different gene variants of a single nucleotide affect nutritional and pharmacological metabolism and gene test results can therefore guide the diet and medication. SNPs are tested either by sequencing, gene arrays or quantitative polymerase chain reaction (qPCR). In this thesis the SNPs were tested by using 132 different TaqMan probes and the SmartChip qPCR platform. 20 participants donated two buccal swab samples and one dried blood spot (DBS) card. Buccal swabs were extracted using two different methods. DNA was also sent to a reference laboratory to be analysed by gene arrays and two participants also sent their DNA samples to two direct-to-consumer (DTC) commercial gene test companies. Low concentration buccal swab samples (<3 ng/µl) produced mismatches whereas DBS samples had high genotyping accuracy even at low DNA concentration. For buccal swabs there was a correlation between the DNA concentration and qPCR call rate (p<0.0001) but not for DBS samples (p>0.05). The allele separation of one TaqMan assay was not sufficient between minor homozygote and heterozygote clusters. For other 131 TaqMan assays excluding the low DNA concentration swab samples, the reproducibility rate was 99.97 %. The reproducibility rate with the DTC companies was 98.36 %. The lower reproducibility rate emphasizes the importance of the manual review of the genotyping raw data, which is not possible with huge sequencing or microarray SNP datasets. SNPs are found on various genetic structures which leads to inconsistent genotyping data that is difficult to analyse with a single algorithm. The SmartChip system combined with the TaqMan assays is a highly reproducible SNP genotyping method when the DNA concentration of the samples is high and the results are manually reviewed.

Trichoderma reesei is an industrially widely utilized filamentous fungus currently used mainly for production of cellulases and xylanases. As T. reesei is a well-known, powerful and robust protein production organism, it is also being developed for heterologous expression of food protein and various enzymes by substituting the main cellulase coding sequence with the sequence of the produced protein. Transcription factor -based regulation of cellulase production in the fungus is well known, whereas thus far another member of Sordariomycetes, Neurospora crassa, has been the main model organism for noncoding RNA (ncRNA) studies in filamentous fungi. To find new ncRNA-derived regulatory mechanisms involved in cellulase expression in T. reesei, previously characterized potential micro-RNA-like RNAs (milRNAs) as well as other potential ncRNAs near cellulolytic pathway genes were deleted using CRISPR-Cas9. These candidates were based on existing total RNA expression data aligned with the Trichoderma reesei genome (Trire v2.0). The effects of these deletions on cellulase production, protease production, total protein production, and growth were analyzed by well-established methods. One purified deletion strain with unchanged growth in cellulase-inducing conditions (2% lactose) was established. Interestingly, this strain displayed low protein production (16%) together with abolished activity of cellulases (3%) and proteases (5%) compared to the parent strain. These findings warrant further studies on the nature of the deleted area, which could perhaps be an unannotated exon based on the RNA expression data. Another strain with a different deletion had abolished cellulase activity with otherwise largely unchanged properties, and all except for one of the analyzed deletion strains had significantly (p<0.05) diminished cellulase activity despite largely unchanged growth. Further investigation of the deleted areas is needed to elucidate the functions of these regions in more detail than was possible in the scope of this work. Seven potential noncoding RNAs were deleted in this work with interesting results that are likely to be useful in improvement of T. reesei production strains in the future.