Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

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No description available.

One of the oldest and most exciting questions in science is: are we alone in the universe? During the last four billion years of Earth’s history, countless organisms have inhabited almost every environmental niche on the planet, from the deepest sea to driest deserts. However, so far no extraterrestrial life has been found. Studying the propensity for life on our neighboring planet, Mars,helps us understanding its potential for past and present life, and guides future missions. Liquid water is a prerequisite for life as we know it and recently, evidence of transient night time liquidbrines on the surface of present day Mars have been theorized. These brines may be hyper-salinewith high ionic strengths and varying pH-values. Halobacterium salinarum is an extremophilic (saltloving) halophilic archaeon whose natural habitat includes hyper-saline brines, desiccating conditions and exposure to high fluences of solar UV radiation. Herein, we report the response of Hbt.salinarum following exposure to simulated Martian conditions, with regard to survival and DNAintegrity. The simulated conditions include the synthetic Martian Brine Analogues (MBAs), diurnalnocturnaltemperature cycling, prolonged desiccation and Mars-like solar UV (200-400 nm) radiation.We also addressed the prolific space hardware contaminant, Bacillus subtilis whose endospores show substantial resistance against space conditions. The ambition was to investigate potentia linterplanetary forward contamination by Hbt. salinarum, should it have bacterial spores available as nutrients in the Martian brines. Halophiles are some of our best candidates for studying unicellular life on Mars and other bodies where liquid water is also stabilized by high salt concentrations.Moreover, Hbt. salinarum was able to survive over one month in the Martian brines, albeit with growth limited to one particularly hospitable brine. It displayed survival in the brines at relevant temperatures and with diurnal-nocturnal cycling but only when first desiccated to remove preventwater crystal formation. The radiation resistance was highly dependent on the choice of brine inwhich Hbt. salinarum was confined and desiccated. Even in the hospitable brines, the halophile lost over 90% of its viable population following irradiation equal to one Martian day, in our experimentalsetup. The inter-brine difference in DNA fragmentation following irradiation confirmed the differencein survival. Hbt. salinarum was subsequently unable to digest B. subtilis endospores for nutrient exploit and responded no differently than when nutrient-deprived. Surprisingly, the addition of otherwise available nutrients in the brines caused a hurried decrease in survival, with the exception of the hospitable brine. Despite its extremophilic and polyextremotolerant character, Hbt. salinarumis unlikely to survive, not to mention thrive, in a combination of all tested stressors.

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No description available.

No description available.

While the Canadian food supply is among the healthiest in the world, almost 4 million (1 in 8) Canadians are affected by food-borne illnesses, resulting in 11,600 hospitalizations and 238 deaths per year. Microbial pathogens are one of the major causes of foodborne sicknesses that can grow in food before or following packaging. Food distribution is an important part of the food processing chain, in which food supplies are at a higher risk of contamination due to lack of proper monitoring. Among myriad of research around biosensors, current devices focusing on packaged food monitoring, such as leakage indicators or time temperature sensors are not efficient for real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging and capable of tracing the presence of pathogens, are of great interest to ensure food safety. This work involves developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which report the presence of a target bacterium in food or water samples by generating a fluorescence signal that can be detected by simple fluorescence detecting devices. The covalently-attached DNA probes generate the signal upon contact with the target bacteria with as low as 103 CFU/mL of Escherichia coli in meat and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to detecting pathogens on packaged food or drinking bottles, these surfaces have the potential to be used for a variety of other applications in health care settings, environmental monitoring, food production chain, and biomaterials like wound dressing. / Thesis / Master of Science (MSc) / Microbial pathogens can grow in food following packaging and preceding consumption. Current biosensors are not efficient for post-packaging real-time food monitoring without separating the sample from the stock. Packaged food such as meat and juice are directly in touch with the surface of their containers or covers. Therefore, real-time sensing mechanisms, installed inside the food packaging, tracing the presence of pathogens, are much useful to ensure the food safety. Here we report on developing thin, transparent, flexible and durable sensing surfaces using DNA biosensors, which generate a fluorescence signal in the presence of a target bacterium in food or water samples. The covalentlyattached DNA probes can detect as low as 103 CFU/mL of Escherichia coli in meat, sliced apple and apple juice. The fabricated sensing surfaces remained stable up to several days under varying pH conditions (pH 5 to 9). In addition to pathogen monitoring in packaged food or drinking bottles, these surfaces are promising for a variety of other applications in health care settings, environmental monitoring, and biomaterials like wound dressing.

Hintergrund: Die Pathogenese nicht-familiärer kolorektaler Neoplasien ist heute noch nicht bekannt. Die Besonderheiten der Epidemiologie der Erkrankung sprechen für die Beteiligung von Umweltfaktoren, wie sozioökonomischer Faktoren, der Ernährung oder der bakteriellen Flora des Darmes. Eine intrazelluläre, von E.coli dominierte Flora wurde in Kolonbiopsien dieser Patienten beschrieben. Methoden: Es wurden Virulenzfaktoren von Escherichia coli untersucht, die aus gewaschenen koloskopischen Biopsien von 43 Patienten mit kolorektalen Adenomen und Karzinomen isoliert worden waren. 100 Stämme wurden mittels PCR auf Gene für folgende Virulenzfaktoren untersucht: s-Fimbrien (sfa), pyelonephritisassoziierter Pilus (pap), Hämolysin A (hlyA), hitzestabiles und -labiles Toxin (ST, LT, EAST), Intimin (eae), Verotoxin (stx), Invasionsplasmid (ipa), cytolethal distending toxin (cdt) und cytotoxic necrotizing factor 1 (cnf1). Für die Kontrollgruppe wurden E.coli aus Biopsien von 55 Patienten mit chronisch-entzündlichen Darmerkrankungen (CED) und unspezifischer Kolitis, von 16 Patienten mit Colon irritabile (IBS) und aus Stuhlproben von 29 gesunden Probanden isoliert und untersucht. Ergebnisse: Bei 69% der Patienten mit kolorektalen Karzinomen und bei 58% der Patienten mit kolorektalen Adenomen wurde mindestens einer der Virulenzfaktoren gefunden, dagegen nur bei 25 bis 39% der IBS- und CED- Patienten sowie der gesunden Probanden (p / Background: Pathogenesis of non-hereditary colorectal neoplasia is poorly understood. The differences in regional incidence indicate an influence of environmental factors, as socio-economic conditions, nutrition and intestinal flora. An intracellular flora with a predominance of Escherichia coli in colon biopsies has been described in these patients. Methods: We studied virulence factors of Escherichia coli isolated from washed colonoscopic biopsies of 43 patients with colorectal carcinoma and adenoma. 100 strains of E.coli were isolated and used for detection of a broad range of virulence genes by PCR encoding: s-fimbriae (sfa), pyelonephritis-associated pili (pap), hemolysin A (hlyA), heatstable and heatlable toxins (ST, LT, EAST), verotoxin (stx), invasionplasmidantigen (ipaH), intimin (eae), cytolethal distending toxin (cdt) and cytotoxic necrotizing factor 1 (cnf1). E.coli from biopsies of 55 patients with inflammatory bowel disease (IBD) and non-specific colitis, of 16 patients with irritable bowel syndrom (IBS) and from stool samples of 29 healthy individuals were isolated and examined as controls. Results: The prevalence of virulent strains bearing at least one of the tested genes was 69% in colorectal carcinoma and 58% in colorectal adenoma, but only 25 to 39% of IBD and IBS patients and healthy individuals (p

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>