Browsing by discipline "Biotechnology (EYT)"

Plant biomass consists largely of polymeric compounds of which diverse polysaccharides are the main components. Ferulic acid is a ubiquitous phenolic phytochemical in plant cell wall and forms the linkage between plant cell wall polymers. Therefore it is a major aspect in the recalcitrance of cell wall against microbial attack. Ferulic acid esterases (FAEs) are hydrolytic enzymes which participate in plant biomass degradation by removing ferulic acid from the polysaccharides in order to weaken the integrity of the cell wall. By using phylogenetic gene prediction strategy, three putative FAE gene models have been detected from the genome of the ascomycete fungus Aspergillus niger. The codon optimized putative FAE encoding genes have been synthetized for heterologous production in Pichia pastoris GS115. In this work, these three FAEs of A. niger, i.e. FAE796, FAE807 and FAE809, were produced in P. pastoris and their biochemical properties were characterized. The properties included substrate profiling, thermostability, pH optimum and solvent tolerance of the recombinant FAEs. The three A. niger FAEs were successfully produced in P. pastoris resulting as approximately 57 kDa molecular mass proteins. Substrate profiling was performed by using a set of 11 synthetic FAE model substrates and substrates for tannase and lipase activity. FAE796 and FAE809 preferred methoxy substrates, and thus were likely to belong to type A class of FAEs. FAE807 had activity towards a wider range of substrates including methyl sinapate, methyl cinnamate, chlorogenic acid and para-nitrophenyl ferulate, suggesting it to belong to type C class of FAEs. In addition, FAE807 had tannase activity which is a novel property described among the FAEs studied so far. The optimal temperature for FAE796, FAE807 and FAE809 were +37 °C, +55 °C and +55 °C, respectively. FAE809 was the most thermostable enzyme, and retained half of its activity up to +60 °C for 60 min. The studied FAEs were most active at pH 4.0-5.0. FAE809 was relatively stable towards the studied solvents retaining 70%-91% of its activity after solvent treatment.

Long QT syndrome, LQTS, is a congenital or acquired cardiac disorder characterized by prolonged cardiac repolarization phase. It is observed as a prolonged QT period in electrocardiodiagraph and can cause life-threatening specific ventricular tachycardia, torsades de pointes. Hundreds of mutations in 15 genes (LQT1-15) are linked to congenital LQTS. Worldwide prevalence of congenital LQTS gene mutations is from 1:2000 to 1:5000. However, the prevalence in Finland is much higher due to four founder mutations that alone occur in one out of 250 individuals. Acquired LQTS is often drug-induced and the most common cause for the withdrawal of drugs on the market. Carriers of LQTS mutations are more susceptible to acquired LQTS than normal population. LQTS-specific cardiomyocytes can thus provide a thorough model for drug cardiotoxicity screening, better insight into disease mechanisms and assist in drug development. This thesis was a part of a bigger project concentrating on validation of LQT2-specific cell lines that could be used for the purposes mentioned above. Induced pluripotent stem (iPS) cell technology enables creation of disease-specific pluripotent stem cell lines, which can be differentiated into any cell type. In this thesis, two LQT2-specific iPS cell lines derived from a clinically symptomatic 44-year-old female were used. She is heterozygous for Finnish founder mutation L552 in KCNH2 gene, which encodes the α-subunit of the cardiac rapidly activating potassium rectifier channel. iPS cells were first verified to express pluripotency markers and to form embryoid bodies containing all germ layers. iPS cells were then differentiated into cardiomyocytes by culturing them with END-2 cells and mechanical beating of the cardiomyocytes was assessed from video recordings. Single LQT2-specific cardiomyocytes showed LQT2-related phenotype in vitro with 43% of single LQT2 cardiomyocytes showing abnormal beating patterns and prolonged contraction time. This phenotype was rescued in LQT2-specific cardiomyocyte clusters. Finally, the expression ratios of wild type and mutated KCNH2 alleles were compared between cardiomyocytes derived from the female and her son, a carrier of the same mutation but with asymptomatic phenotype. Cardiomyocytes from both individuals expressed KCNH2 alleles with the ratio between 1:2 and 1:1 (wt:mut), thus allelic imbalance does not explain differences in the clinical phenotypes. All in all, the results of this thesis suggest that after further validation, mainly electrophysiology studies, these cell lines are most likely suitable to be applied for disease modeling, cardiotoxicity screening and finding new therapies for LQT2.

Microbial cellulases, e.g. cellobiohydrolases, are able to degrade cellulose and lignocellulosic biomass to smaller glucose-containing monomers and oligomers. Cellulases are often multi-domain enzymes comprised of different protein domains (i.e. modules), which have different functions. The main two components, which often appear in cellulases, are the cellulose-binding module (CBM) and the catalytic domain. The CBMs bind to cellulose, bringing the catalytic domains close to their substrate and increasing the amount of enzymes on the substrate surface. The catalytic domain performs the cleavage of the substrate, e.g. in the case of cellobiohydrolases hydrolyses or “cuts” the crystalline cellulose chain into smaller soluble saccharides, mainly cellobiose. Unlike aerobic fungi, which utilize free extracellular enzymes to break down cellulose, anaerobic microbes often use a different kind of strategy. Their cellulases are organized and bound to the cell surface in a macromolecular protein complex, the cellulosome. The core of the cellulosome is formed of a scaffolding protein (the scaffoldin) consisting mainly of multiple consecutive cohesin domains, into which the catalytic subunits of enzymes attach via a dockerin domain. This creates a protein complex with multiple different catalytic domains and activities arranged in close proximity to each other. Dockerins and cohesins are known to bind each other with one of the strongest receptor-ligand -pair forces known to nature. Dockerin containing fusion proteins have also been successfully combined in vitro with proteins containing their natural counterparts, cohesins, to create functional multiprotein complexes. In this Master’s thesis work the goal was to 1) produce fusion proteins in which different CBMs were connected to dockerin domains, 2) combine these fusions with cohesin-catalytic domain fusion proteins to create stable CBM and catalytic domain containing enzyme complexes, 3) to characterize these enzyme complexes in respect of their thermostability and cellulose hydrolysis capacity and 4) to ultimately create a robust and fast domain shuffling method for multi-domain cellobiohydrolases (CBH) to facilitate their faster screening. The hypothesis of the experiments was that different CBMs fused with a dockerin domain and the cellobiohydrolase catalytic domain fused with a cohesin domain could be produced separately and then be combined to produce a functional two-domain enzyme with a dockerin-cohesin “linker” in between. In this way time and work could be saved because not every different CBM- catalytic domain -pair would have to be cloned and produced separately. Several CBM-dockerin fusion proteins (in which the CBM were of fungal or bacterial origin) were tested for expression in heterologous hosts, either in Saccharomyces cerevisiae or Escherichia coli. The purified proteins were combined with a fungal glycoside hydrolase family 7 (GH7) cellobiohydrolase-cohesin fusion protein produced in S. cerevisiae. The characterization of the catalytic domain-CBM -complexes formed through cohesin-dockerin interaction included thermostability measurements using circular dichroism and activity assays using soluble and insoluble cellulosic substrate. The results were compared to enzyme controls comprising of the same CBM and catalytic domain connected by a simple peptide linker. The results showed that the cohesin-dockerin –linked cellobiohydrolase complex performed in the cellulose hydrolysis studies in a similar manner as the directly linked enzyme controls at temperature of 50˚C and 60 ˚C. At temperatures of 70 ˚C the complex did not perform as well as the control enzymes, apparently due to the instability of the dockerin-cohesin interaction. The thermostability measurements of the enzymes, together with the previously published data supported the hydrolysis results and this hypothesis. The future work should be aimed at enhancing the thermostability of the cohesin-dockerin interaction as well as on verifying the results on different cellulase fusion complexes.

The aims of this study were to use the heterologously expressed laccase in Pichia pastoris as oxidative biocatalysts for the degradation of BPA and to find out natural mediators that could assist this laccase to degrade BPA efficiently and environmental friendly. In addition, removal of the estrogenic activity of BPA by the white rot fungus Physisporinus rivulosus cultures and the role of its laccase 2 in the BPA disapperance was also focused. Firstly, the removal of BPA’s estrogenic activity by the white rot fungus Physisporinus rivulosus was confirmed in cultures both with and without the fungal mycelium. Next, the recombinant laccase 2 (rLac2) played a role in the BPA disapperance. At the similar laccase activity level, removal of BPA’s estrogenic activity was done more efficiently in the fungal cultures than in the cell-free enzymatic treatments. Metabolites present in the fungal cultures could possibly act as natural mediators that enhance the removal of BPA. In addition, combination of nine laccases present in the fungal cultures could possibibly enhance the degradation of BPA. Finally, none of the ten mediatiors used was found to act as an efficient rLac2 enhancer in degrading BPA. Degradation of BPA was followed using a bioreporter system. The yeast Saccharomyces cerevisiae has been genetically modified in order to express the estrogen receptor alpha and produce a bioluminescent signal upon contact with estrogenic substances such as BPA. This bioreporter system has been further developed to specifically detect the estrogenic activity of bisphenol A and to be used in a high-throughput manner.

The current multidisciplinary interests on human intestinal microbiomes have stimulated large scale research initiatives, involving collection and processing of up to thousands of fecal samples within a single study. Hence, there is a need for high throughput protocols that are cost-efficient and validated for their performance to ensure that the relative abundance of different bacteria, the main outcome of microbiota studies, is not biased due to technical artefacts originating from sample processing. Infant’s microbiota colonization is one of the central research areas in human microbiome research because of the long-lasting and profound health implications of the pioneering microbes. This experimental study aimed to develop and validate a high throughput fecal sample collection and processing system for microbial DNA extraction operating in 96 well format. This newly developed method was used to extract DNA from 647 fecal samples collected from mother-infant pairs within a clinical study that will study the effect of antenatal antibiotic prophylaxis on infant’s gut microbiota development. A subset of 28 mother-infant pair samples (14 from each antibiotic and non-antibiotic groups) were selected to study the prevalence of a probiotic bacterium, Lactobacillus rhamnosus GG (LGG), among infants and their mothers longitudinally from birth to 3 months by using species-specific PCR amplification method targeting sortase C gene. In addition, the prevalence of L. rhamnosus GG in 3-month-old infants was compared between the above samples and those (n=30) collected in another clinical trial conducted ~10 years earlier. From extensive testing and validation, an efficient high throughput system for fecal sample collection and processing for extraction of microbial DNA in 96 well format was established. Tests were performed to validate the performance of a) fecal sample collection system b) commercial, readymade bead beating tubes for bacterial cell lysis and c) selfmade wash buffers as part of the automatic DNA purification system. Performance was evaluated based on the quality and quantity of the resultant DNA. We show that this new fecal processing system can yield high quality microbial DNA from 96 fecal samples within ~6 hrs. Based on the ratios of dominant gram-positive and gram-negative bacteria evaluated using PCRs and next generation sequencing, the new DNA extraction method resulted into similar microbiota composition than the previously validated manual DNA extraction method. However, the DNA yield per sample was markedly lower due to the lower input volume of the sample. Based on the sortase C gene PCR tests, the prevalence of LGG was similar (~60%) among 3-month-old children in both clinical studies conducted ~10 years apart, although false negatives among the recent samples due to the low amount of DNA cannot be excluded. Following the temporal pattern of colonization, we observed no evidence for the transfer of LGG at the time of birth from the mother to her child, instead the infants became positive for LGG typically between 1-3 weeks after birth. The carriage of LGG seemed to be dependent on their diet. During this project, we found out that the PCR method employed for detection of LGG was not fully specific for this strain, and hence a more specific qPCR assay was developed.

New alternative feedstocks are needed for biofuel production to fulfil the growing demand in the coming years. The industry is moving away from second-generation biofuels, produced from food and feed crops, to using waste streams from industrial processes. An abundant, cheap and attractive waste stream for processing in Europe is the pectin-rich pulp from sugar beet processing and fruit juice industry. Sugar beet pulp is particularly rich in D-galacturonic acid and arabinose, but neither are naturally used by the yeast Saccharomyces cerevisiae, which would be an interesting candidate for the microbial fermentation of the biomass. S. cerevisiae is one of the most used organisms in the industrial biotechnology, and methods for the genetic engineering of the organism are highly developed. To overcome the natural limitations of the yeast for D-galacturonic acid fermentation, the metabolic pathways present in other organisms could be integrated in the yeast genome. Two bacterial and one fungal pathway are known to convert D-galacturonic acid into metabolites of the yeast glycolytic and ethanol fermentation pathways, and are thus considered promising for engineering in yeast. A major engineering challenge in integrating the fungal pathway in yeast is the redox imbalance caused by the two NADPH-specific reducing enzymes. The aim of this thesis was to review the potential of different D-galacturonic acid pathways for yeast fermentation. S. cerevisiae is a well-characterised organism for heterologous protein expression, but at times functional expression of foreign proteins is not achieved. One approach to study the pathways was to clone and express enzymes of the bacterial isomerase and dehydratase pathways in S. cerevisiae, and to test their activity in culture lysates. In addition, to overcome the redox imbalance in the eukaryotic pathway, two approaches were used to obtain an NADH-spesific D-galacturonic acid reductase. First, a mutant library of the Trichoderma reesei gar1 reductase was designed with the structure-guided cofactor specificity reversal tool CSR-SALAD. An automated high-throughput screening method for expression in Escherichia coli was developed, and the library was screened for enzymatic activity. The second approach was to try to identify the sequence for the characterised NADH-utilising reductase from the single-cell algae Euglena gracilis. A cDNA library of the algae was made and screened with PCR and in vivo methods. The reductase uxaB of the isomerase pathway and dehydrogenase kduD of the dehydratase pathway were functionally expressed in S. cerevisiae, with specific activities of 1.1 µmol min-1 mg-1 and 0.22 µmol min-1 mg-1 , respectively. The enzymes dehydratase uxaA and isomerase kduI did not exhibit activity in activity assays. The galurD of the dehydratase pathway was expressed in E. coli, and the purified enzyme was successfully used to convert D-galacturonate to 5-keto-4-deoxy galacturonate. The approaches to change the cofactor specificity of the NADPH-specific reductase of the eukaryotic pathway did not lead to a discovery of a NADH-specific enzyme. More research is needed for engineering active enzymes for S. cerevisiae expression and constructing a fully functional D-galacturonic acid pathway for feasible D-galacturonic acid fermentation.

Bacteria can communicate with each other using phenomenon called quorum sensing (QS). In QS the bacteria produce and release small signaling molecules which they use to communicate. Bacteria use QS in situations where it is beneficial to act on population level. QS has an important role e.g. in the formation of virulence factors and biofilms. There are several different QS systems. Gram-negative bacteria use i.a AI-1, AI-2, AI-3, and CAI-1 systems to communicate. All QS systems are based on the accumulation of signaling molecules when the bacterial concentration increases. When the concentration of signal molecules reaches the threshold level, the system activates. The activation of the signaling system then activates the expression of the genes controlled by the QS system. AI-2 signaling is assumed to be universal. That means that bacteria can use AI-2 signaling system in interspecies communication. In AI-2 signaling bacteria produce and release 4,5-dihydroxy-2,3-pentanedione (DPD) which works as a signaling molecule in the AI-2 system. Escherichia coli and Salmonella typhimurium use an ATP binding cassette ABC-type transporter to transport DPD molecules into the cell where LsrK kinase phosphorylates the DPD molecules. The phosphorylated DPD molecules bind to the LsrR regulator protein which acts as a suppressor of the lsr operon. The binding of the phosphorylated DPD molecules releases the LsrR from the lsr promoter region enabling the expression of the lsr genes. In Vibrio harveyi the surface proteins LuxP and LuxQ form a protein complex that recognizes DPD molecules. When the DPD concentration increases, the LuxPQ complex transform from kinase to phosphatase and the reaction chain, where LuxU phosphate transfer protein transfers a phosphate group from LuxO regulator protein, activates. The dephosphorylation of of LuxO releases the LuxR transcription factor and activates the expression of QS controlled genes. The aim of this thesis was to optimize two assays which can be used to screen for compounds that disrupt AI-2 signaling. The first assay was a bioreporter based assay where V. harveyi BB120 bioreporter strain was used. The second assay was protein based LsrK assay where the LsrK activity was monitored using assay kit which measures the concentration of ATP or ADP. The concentrations of bacteria, LsrK, and DPD used in the assays were optimized. The dimethyl sulfoxide (DMSO) tolerance of both assays were tested, the stability of the kits used in the LsrK assays was tested and the reaction buffer for the LsrK assay was selected from the two tested buffer options. The selected bacterial concentration for the V. harveyi BB120 assay was 100000 CFU/ml and DPD concentration 1 µM. The selected enzyme concentration for the LsrK assay was 300 nM and DPD concentration 300 µM. The tested DMSO concentrations had no effect on the kit measuring ATP but the highest concentrations tested had a small effect on the kit measuring ADP. A buffer containing triethanolamine, magnesium chloride, and bovine serum albumin was selected as the reaction buffer for the LsrK assay. Using the optimized LsrK assay, a screening was performed for a synthesized compound library. None of the compounds showed any LsrK inhibiting activity. The optimized assay was also used to make dose-response experiment to one LsrK inhibiting compound, named FIMM000642, which was found in a separate screening. The FIMM000642 dose-response as-say was also done against glycerol kinase to see if the compound would inhibit another enzyme from the same protein family or if the compound was a specific inhibitor to LsrK. FIMM000642 inhibited also the activity of glycerol kinase.

Cellulose is a major component of plant biomass that can be used to produce bio-derived materials, chemicals, and fuels. Whereas the conversion of cellulose to chemicals and fuels is limited by the surface functionality of cellulose fibers due to their highly compact structure. A group of non-catalytic proteins including expansins, loosenins and cerato-platanins are known to alter cellulose fiber structures, presumably by breaking hydrogen bonds between cellulose microfibrils. This makes them interesting targets for the chemical and biophysical modification of plant polysaccharides. In this work, seven non-catalytic target genes were selected from the transcriptome of Phanerochaete carnosa. Pichia pastoris was transformed with expression constructs harboring the respective genes under control of the AOX1 promoter. Protein expression was analyzed by colony blot assay and small-scale expression in liquid media. Two cerato-platanins (CP1, CP4) and 3 loosenin-like proteins (LOOL7, LOOL9 and LOOL12) were successfully produced in large-scale. The identity of CP1, LOOL7, LOOL9 and LOOL12 was confirmed by MALDI-TOF MS analysis. The proteins showed no hydrolytic activity when tested on carboxymethyl cellulose, xylan or glucomannan. Defibrillation assay results of CP1, CP4, LOOL7 and LOOL9 suggested that the non-catalytic proteins lack specific weakening effect on cellulose filter paper. Considerably high protein blank absorbance of LOOL7 indicates possibility for self-assembling abilities of the protein. Incubation of filter paper with CP1 seemed to improve the hydrolytic action of cellulases on Avicel and filter paper, as shown by complementation assays. The effect of LOOLs on enzymatic cellulose hydrolysis varied over the time course of different experiments. Most notably, LOOL7 and LOOL12 were able to increase the reducing sugar release from Avicel and filter paper by cellulase at different time points. The maximum increase of cellulose conversion achieved by LOOL12 was 30% after 10 min when non-catalytic proteins and Cellic CTec2 were added simultaneously. LOOL7 showed highest conversion improvement of 21% after 6 h pre-incubation assay with filter paper. Upon prolonged pre-incubation (72 h), significant improvement of filter paper hydrolysis was noticed for CP4 and CP1, while LOOLs unable to enhance the hydrolytic activity of the tested cellulase mixture. For the first time, a comparative study of cerato-platanins and loosenin-like proteins was carried out and the findings presented in this thesis suggest that especially CP1 can be a promising accessory protein for efficient bioconversion of cellulose. Nevertheless, a deeper understanding of the structure and function of these non-catalytic proteins will be helpful in determining their potential biotechnological applications.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease in the western world. The human intestinal microbiota possesses enormous metabolic and immunomodulatory capabilities, and together with increased intestinal permeability, changes in the microbiota have been linked to the development of NAFLD. However, human studies so far have yielded contradictory findings regarding the compositional microbiota changes and provided little mechanistic understanding due to the predominance of cross-sectional studies. The aim of this study was to study human intestinal microbiota and gut permeability in NAFLD. Real-time PCR was employed to quantify the key intestinal bacterial groups in overweight or obese subjects with (n = 12) and without (n = 19) NAFLD, and in response to hypercaloric overfeeding, where participants were provided with three compositionally distinct diets to temporarily increase liver fat. In addition to the comparative analysis, the microbiota results were correlated to serum markers of intestinal permeability and metabolic endotoxemia, as well as clinical parameters related to NAFLD. The results show that host lipid metabolism and the gut microbiota, specifically Bacteroidetes and Clostridium cluster XIVa, are firmly intercorrelated. Bacteroidetes were found to be less abundant in subjects with NAFLD and correlate negatively with liver fat and serum triglycerides at baseline. Clostridium cluster XIVa, a dominant Firmicute group, was positively associated with serum triglycerides and pro-inflammatory markers but negatively with intestinal permeability. The relative abundance of Bacteroidetes as well as the markers of metabolic endotoxemia changed significantly in response to overfeeding, while no diet-induced systematic effects were found in Clostridium cluster XIVa, total bacteria, Escherichia coli group, Bifidobacterium or gut permeability. Our results based on a targeted microbiota analysis suggest that the role of the intestinal microbiota and gut permeability on triggering metabolic disarrangement and NAFLD in humans is inferior to other stimuli, such as diet.

Antibiooteille vastustuskykyisistä eli resistenteistä bakteereista on tullut maailmanlaajuinen ongelma, joka on vakavuudeltaan verrattavissa pandemioihin. Tautia aiheuttavien bakteerikantojen vastustuskyky vaikeuttaa infektioiden hoitoa ja voi aiheuttaa pahimmillaan jopa kuolemantapauksia. Bakteerien antibioottivastustuskyky on antibioottiresistenssigeeneihin perustuva ominaisuus, joka on yleistynyt antibioottien käytön seurauksena sekä tautia aiheuttavilla bakteereilla että vaarattomilla ympäristöbakteereilla. Erityisen ongelmallisia ovat liikkuvissa geneettisessä elementeissä sijaitsevat antibioottiresistenssigeenit, joissa on DNA:n aktiiviseen siirtymiseen vaadittavia geenejä. Liikkuvissa geneettisissä elementeissä sijaitsevat resistenssigeenit siirtyvät erittäin tehokkaasti bakteerien välillä, jolloin resistenssigeenien leviäminen ympäristöbakteereilta tautia aiheuttaville bakteereille aiheuttaa maailmanlaajuisesti oleellisen riskin ihmisten terveyden kannalta. Maisterintutkielmassani kehitettiin IPCR:ään (engl. inverse PCR) pohjautuva menetelmä antibioottiresistenssi-geenejä ympäröivien geenien tutkimiseen. Antibioottiresistenssigeenejä ympäröivien alueiden avulla voitiin päätellä, sijaisivatko resistenssigeenit liikkuvassa geneettisessä elementissä, ja sitä kautta arvioida antibioottiresistenssigeenien leviämisriski. Liikkuvissa geneettisissä elementeissä on antibioottiresistenssiresistenssigeenien lisäksi muita kullekin liikkuvalle geneettiselle elementille tyypillisiä geenejä, jotka voitiin monistaa käyttämällä kehitettyä IPCR-menetelmää. Menetelmää käytettiin kalanviljelylaitoksen bakteerien sul1- ja tetM-resistenssigeenejä ympäröivien geenien tutkimiseen. IPCR:ää ei ole aikaisemmin sovellettu liikkuvien geneettisten elementtien ja antibioottiresistenssigeenien tutkimiseen ympäristönäytteillä. IPCR:ää ja uuden sukupolven PacBio-sekvensointimenetelmää käyttämällä onnistuttiin monistamaan ja sekvensoimaan useita sul1- ja tetM-geenejä sisältäviä liikkuvia geneettisiä elementtejä, mikä osoitti, että menetelmä sopii antibioottiresistenssigeenien liikkuvien geneettisten elementtien tutkimiseen ympäristössä. Kaikki sul1- ja tetM-geenit sijaitsivat liikkuvissa geneettisissä elementeissä, minkä vuoksi geeneillä on suuri siirtymisriski kalankasvattamolla esiintyviltä ympäristöbakteereilta ihmisille tai kaloille tautia aiheuttaville bakteerikannoille. tetM-geenit sijaitsivat Tn916-tyyppisissä transposoneissa tai sul1-geenin kanssa samassa integronissa. sul1-geenit sijaitsivat puolestaan integroneissa, joissa oli lisäksi muita resistenssigeenejä, ja myös geenejä jotka aiheuttavat vastustuskykyä terveydenhuollossa tärkeille antibiooteille, joita ei käytetä kalanviljelyssä. Yksi sul1:n kanssa samasta liikkuvasta elementistä löytyneistä geeneistä oli mahdollisesti uudentyyppinen blaoxa-β-laktaamiantibioottiresistenssigeeni. Uuden mahdollisen resistenssigeenin löytyminen osoittaa, että IPCR soveltuu hyvin myös uusien liikkuviin elementteihin liittyneiden resistenssigeenien etsimiseen. Maisterin tutkielmassani kehitetty IPCR-pohjainen menetelmä, joka perustui perinteisiin molekyylibiologisiin tekniikoihin eikä vaatinut kalliita reagensseja tai laitteita, osoittautui käyttökelpoiseksi ympäristössä esiintyvien antibioottiresistenssigeenien siirtymisriskin arvioimiseen. Menetelmää voitaisiin jatkossa käyttää edullisesti ja helposti antibioottiresistenssigeenien ja -geenejä sisältävien liikkuvien geneettisten elementtien monitorointiin ympäristössä, myös silloin kun antibioottiresistenssigeenit eivät vielä ole levinneet sairaalaympäristöön. Tällöin ihmisen toiminta voi vielä vaikuttaa haitallisten antibioottiresistenssigeenien leviämiseen tautia aiheuttaville bakteereille.