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- PostA CRISPR approach to manipulating NK cell receptor ligand expression(2018) Kristensson, Linnea; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringNatural killer (NK) cells are important innate lymphocytes in the immune system and have been considered to be vital in fighting cancer, virus infections as well as regulating immune responses. The activity of NK cells is regulated via a complex signaling between inhibitory and activating receptors. Ligands for most of the ac-tivating receptors have been discovered but no endogenous ligand for one of the important natural cytotoxicity receptors, NKp46 has been identified. Therefore, the long term aim of this thesis project was to define ligand candidates for the acti-vating receptor NKp46 with intermediate aims to manipulate expression of NK cell activating receptor ligands in the K562 cell line using a CRISPR approach. The result from the performed cytotoxicity assays demonstrated the importance of the activating receptors NKp30 and DNAM-1 in elimination of K562 cells and the expression of the complementary ligands NCR3LG1, PVR and NECTIN2 was confirmed using RT-qPCR. The successful creation of a Cas9-expressing K562 cell line was demonstrated, including verification of Cas9 protein expression using both flow cytometry and western blot. Following that, a constructed plasmid with the gRNA for the B7-H6 gene, NCR3LG1, was generated and shown to create a cleavage and mutation within the gene. The engineered cell line was ultimately shown to have a reduced expression of the B7-H6 protein, indicating the successful knockout of the NCR3LG1 gene.
- PostA kinetically-constrained FBA-model of the synthesis of aromatic amino acid-derived products in Saccharomyces cerevisiae(2015) Janasch, Markus; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringDuring the last decades the research interest for creating a more sustainable and environmentallyfriendly society and industry has increased dramatically. The employment of icroorganisms to create valuable compounds such as fuels and chemicals from renewable resources has gained high popularity. With directed modifications in the metabolism of these microorganisms, metabolic engineering seeks to optimize the properties of these organisms for an efficient bioprocess to produce valuable compounds. One characteristic of metabolic engineering is the extensive use of computational models to predict the behavior of the metabolism and thereby identifying suitable targets for genetic interventions in an in silico to in vivo progress. Flux Balance Analysis (FBA) is a popular analysis method for metabolic models, as it only requires the underlying stoichiometric network of the metabolism modelled. With FBA the optimal flux distribution, given a certain objective to be maximized, can be calculated with linear programming algorithms. As FBA only takes the stoichiometry and reaction directionality into account, further physiological constraints have to be included in the framework to increase the predictive strength of the simulations. In this thesis, an expanded form of FBA, that includes kinetic enzyme parameters as additional constraints on the system, was used to analyze a metabolic model of the popular industrially-used organisms Saccharomyces cerevisiae, that included, additionally to the native metabolism, also recombinant enzymatic steps to form the plant secondary metabolites resveratrol and naringenin from the aromatic amino acids phenylalanine and tyrosine. Resveratrol and naringenin have been found to be beneficial to human health by offering anti-inflammatory, anti-carcinogenesis and anti-oxidant properties. The kinetically-constrained model was successfully used to estimate the impact of introducing recombinant pathways on the protein pool in the cell as well as to identify the enzymatic steps offering the highest control over the flux towards these products. This might be used in metabolic engineering to estimate the efficiency of metabolic pathways in the context of the whole metabolism form a protein cost point of view.
- PostA platform for Acetyl-CoA synthesis using xylose as feedstock in Saccharomyces cerevisiae(2015) Englund Örn, Oliver; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringGlobal rise in temperature and diminishing oil reserves has stimulated a market of alternative replacements to traditional petroleum based products. An alternative is the use of a bio refinery capable of converting biomass to the normally petroleum based products. The baker yeast Saccharomyces cerevisiae is an attractive cell factory as its existing large-scale infrastructures for bioethanol production. However, it cannot utilize xylose, an otherwise unusable part of the plant biomass, which represents of utmost importance in the bio-refinery development. To also have a strain capable to produce a wide range of products, it could be used as a platform to base a bio refinery upon. Therefore, the aim of this study is to generate platform strains capable of forming acetyl-CoA, an intermediate metabolite in many of the cells metabolic reactions and also for many other industrially relevant bio-chemicals. With this goal in mind, the metabolism of S. cerevisiae was engineered. The genes encoding an isomerase-based xylose assimilation pathway (RTG, XI, XKS), and a phosphoketolase pathway (XPK, PTA), were cloned into the yeast strain CEN.PK113-5D to enable the yeast to take up and convert xylose into acetyl-CoA. The functionality of this synthetic pathway were evaluated for the production of 3-hydroxypropionic acid via introduction of ACC1** and MCR genes into the engineered strains. By characterisation of all the engineered strains on glucose growth we found increase of acetate production in strains with the phosphoketolase pathway expressed, indicating the in vivo activity of this pathway. However, expression of the xylose assimilation pathway through genome integration did not render the strains able to grow on xylose, suggesting the low efficiency of the assembled xylose assimilation pathway. To overcome this adaptive laboratory evolution is recommended.
- PostA Rolling Circle Amplification-Based Methodology for Making Long, SequenceRepeating, DNA Duplexes(2018) Dekoning, Nora; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringIn vitro studies of sequence-specific DNA-protein interactions using techniques where long DNA molecules is needed are currently limited by the size of synthesized DNA molecules, or is restricted to the commercially available DNA. For single molecule DNA imaging techniques using for instance nanochannels confinement, the small sized molecules make the interactions difficult to detect. The commercially available DNA, on the other hand, does not allow any freedom in choosing or changing the DNA sequence. Therefore, a method for producing long DNA molecules containing a sequence of choice would alleviate these limitations and greatly improve the possibility to study DNA-protein interactions. The general concept of this paper was to create long, double-stranded DNA molecules with a sequence that is specifically designed to interact with the protein internal host factor. To make such molecules, three experimental procedures were developed, building on the single-stranded DNA products from a rolling circle amplification (RCA) reaction. The first experimental procedure was based on the presumed perfect annealing of smaller complementary strands to the single-stranded RCA product. The second experiment assumed that single-stranded gaps would be present in the duplex after annealing, hence the addition of a polymerisation reaction. In the third experiment, the RCA was run for 24 hours, to allow double-stranded product to be formed in the RCA. The DNA strands were visualized using fluorescence microscopy, with the goal of studying them and their protein interactions in nanochannels. To be able to use this detection method, an external fluorescent dye, YOYO, is used in the main aim of the project. However, as these types of dyes change the native structure of DNA, an extra aim was to use the fluorescent base analogue tC incorporated into one of the duplex strands, leaving the native structure intact. All three experimental procedures were shown to be capable of producing apparently double-stranded DNA molecules, that were larger than 100 kilo-base pairs albeit with a broad size distribution. This shows that the main aim in terms of procuing DNA molecules has been completed. The tC-containing DNA molecules were not visible under the microscope with the settings used, but appears to be promising.
- PostA Step Toward Personalized Cancer Treatment Simultaneous Detection of Multiple Types of Chemotherapyinduced DNA Damage Using Single Molecule Imaging(2022) Foss, Ebba; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological Engineering; Westerlund, Fredrik; Akwasi Aning, ObedChemotherapy is commonly used to treat cancer today, either alone or more commonly as part of combination therapy. Response to a certain chemotherapeutic agent is highly individual, both in terms of treatment efficacy and the extent to which healthy cells are affected. For several drugs, induced DNA damage provides the main cytotoxic effect, and a method for evaluating this damage could therefore prove a powerful tool in treatment planning. In this thesis, a single molecule imaging approach is used to assess chemotherapy-induced DNA damage, allowing visualisation of damage sites on individual DNA strands. While previous studies have focused on one damage type, or collective damage without distinction between types, a novel modification to pre-existing techniques that allows for this distinction has recently been demonstrated. In this thesis, the alkylating agent temozolomide was used to illustrate how different damage types can be distinguished with a single molecule imaging approach. This is done using repair enzymes associated with different DNA repair pathways. The repair enzymes sequentially incorporate spectrally distinct fluorescent nucleotides at the damage site which are then visualized as fluorescent spots of two different colours on individual DNA molecules. This distinction could be shown with high repeatability in terms of colour ratio. While both enzymes used separately clearly repaired the treated DNA, there appeared to be an overlap when applying them sequentially. This could suggest a problem with enzyme specificity. Further exploration of this issue is needed to verify the feasibility of single molecule imaging for the purpose of simultaneous detection of chemotherapy-induced DNA damage types.
- PostAccelerated shelf life tests of wheat tortillas A study of microbial and textural deterioration in wheat tortilla(2018) Nilsson, Fredrik; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThis study tried to shorten the time required for shelf life studies in product development of wheat tortillas, by developing accelerated shelf life test (ASLT) models for microbial and textural shelf life. ASLTs speed up product degradation by exposure to extreme environmental conditions and in this case were temperature and relative humidity (RH) used. The samples were divided into four storage groups: 3°C and 70% RH, 20°C and 30% RH, 27°C and 80% RH as well as 40°C and 80% RH. Texture was determined with a fold/roll-method, an instrument and by a sensory panel. Microbial concentrations were determined by a laboratory company. Gas composition inside the bags, tortilla pH, water content and water activity were measured to determine possible links to the shelf life. It was not possible to calculate an ASLT model for the microbial growth because of unknown starting number of microbes. The microbial tests however showed that a high temperature and humidity caused the number of bacteria to rapidly increase above what is considered acceptable. Microbial ASLTs thus seem possible, but more tests with a lower detection limit are needed to create a model. To avoid bad taste from nonpathogenic bacterial growth, consumers are recommended to avoid storing the tortillas in high temperature and/or humidity. Textural ASLTs also seem possible in regards of rollability and foldability, but more tests are needed to precisely determine the accelerating factor. Stickiness and translucency seems to deteriorate much slower than rollability and foldability under normal storage conditions. It is therefore suggested to be enough to check that freshly baked tortillas meet the quality requirements of stickiness and translucency. Tortillas seemed to lose textural quality at the same rate, or slower, in refrigerator compared to room conditions. Longer shelf life studies are therefore suggested, to verify if this is the case. If so, storage in a refrigerator could possibly prolong shelf life of wheat tortillas. Changes in the modified atmosphere inside the bags seems to correlate with textural degradation. A study with atmospheric air instead would therefore be interesting as an attempt to determine if it retards textural degradation in wheat tortillas.
- PostAdaptive evolution of Yarrowia lipolytica(2017) Hellgren, John; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringWe need renewable resources to allow sustainable production of fuels. By using lipid accu-mulating yeast, biodiesel can be produced in a sustainable way from resources that were previously not used, for example lignocellulose-based sources such as agricultural waste. However, for this process to be proﬁtable, the tolerance of the yeast needs to be improved. This project aims to improve the tolerance of the oleaginous yeast Yarrowia lipolytica to-wards osmotic and saline stress by using the method adaptive laboratory evolution. This method has previously been shown to be eﬃcient in constructing strains to tolerate new conditions without the need of prior knowledge. After evolving Y. lipolytica for 220 gen-eration in minimal medium containing 1.4 M NaCl, an improved performance in the same medium was observed, along with evolved cross-tolerance towards low pH. This indicates that this adaptive evolution of Y. lipolytica resulted in improved ionic tolerance rather than pure osmotic tolerance. The evolved strains were sent for whole genomic sequencing to ﬁnd out which mutations that caused this phenotype. During this project, two previous CRISPR/Cas9 strategies were combined and adapted for eﬃcient markerless reverse en-gineering. When genome data arrives, this strategy will be used for reconstruction of can-didate mutations to ﬁnd out which mutations are important for the observed phenotype. The gained knowledge from this evolution experiment can later be used for constructing a robust industrial strain that eﬃciently converts lignocellulose-based material to biodiesel, allowing sustainable production of fuels.
- PostAdaptive laboratory evolution of Saccharomyces cerevisiae for increased tolerance towards compounds of industrial interest(2016) Malina, Carl; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringOver the last decades, microbial production of fuels and chemicals has become an increas-ingly attractive alternative to petroleum-based production. This has created a demand cell factories able to produce a wide range of compounds. The yeast Saccharomyces cere-visiae is widely used in biotechnology with successful applications in the production of both bulk and fine chemicals. However, in order to reach the full potential of yeast as a cell factory challanges still remain. These include creating strains tolerant to stress conditions, such as inhibition at the high product titers required in industrial production. Traits conferring these properties are often complex and encoded by several genes. In order to obtain strains with improved tolerance, adaptive laboratory evolution (ALE) is often used. This thesis was part of an ongoing project of ALE for increased tolerance towards compounds with potential industrial applications. The work in this thesis can be divided into two main parts. The first part was screening for tolerance of S. cerevisiae towards four diols and two diamines by microplate cultivation. For both the diols and diamines, clear trends of decreasing fitness as compound concentration was increased was seen. For the diols tested, it seems increasing toxicity correlates with increasing chain length and branching. The second part was characterization of pimelic acid tolerant strains from ALE, by shake flask cultivation and HPLC analysis of metabolites. In addi-tion, the genomes of 21 strains were resequenced. Results from the shake flask cultures showed that, in presence of pimelic acid, most strains have an impaired growth on non-fermentable carbon sources. Furthermore, HPLC analysis of metabolites revealed that glycerol and acetate accumulated during cultivation while ethanol was slowly consumed, implicating a defective respiratory system. Through genome resequencing, in total 47 genes were found to be mutated across all of the evolved strains.
- PostAddition of starch in an airlaid process to the cellulose-based composite DuraPulp(2016) Klein, Julia; Chalmers tekniska högskola / Institutionen för kemi och kemiteknik; Chalmers University of Technology / Department of Chemistry and Chemical Engineering
- PostAntimicrobial Effect on Bacteria in Biofilm and Planktonic state(2018) Linse, Julia; Larsson, Anna; Chalmers tekniska högskola / Institutionen för fysik (Chalmers); Chalmers University of Technology / Department of Physics (Chalmers)The elimination of contaminating bacteria is essential in order to achieve proper wound healing. Bacteria can occur in two different states, as free-living (planktonic) cells or in aggregates adhered to a surface (biofilm). The bacteria behaves differently depending on in which state they occur and their differences and their sensitivity against Chlorhexidine digluconate have been of key interest during this thesis work. To investigate this, time-kill studies have been performed on the two common wound bacteria P. aeruginosa and S. aureus under static and dynamic conditions. The studies were performed on bacteria in both biofilm and in planktonic state. The minimum inhibitory concentration (MIC), the minimum biocidal concentration (MBC) and the minimum biofilm eradication concentration (MBEC) for the antimicrobial compound Chlorhexidine digluconate against the bacteria were used in time-kill studies under static and dynamic conditions. In the static time-kill studies the bacterial were treated with different concentrations of Chlorhexidine digluconate in the range from below the MIC to above the MBC. In the dynamic time-kill studies Chlorhexidine digluconate was added continuously to the bacteria to reach the determined MBC in different times. Results from the experiments showed that P. aeruginosa was less sensitive against treatment of Chlorhexidine digluconate than S. aureus was. There seemed to be persister cells within the P. aeruginosa population during all experiments since they managed to recover from antimicrobial treatment even if the bacterial concentration at a point was below the limit of detection. The experiments also showed that killing of P. aeruginosa seemed to be Cmax-driven, which means that a high concentration Chlorhexidine was needed to be delivered fast to the bacteria to achieve the best rate of killing. For S. aureus the killing seemed to be AUC-driven which means that a lower amount of the antimicrobial could be used but the bacteria needed to be exposed for a longer time. When the two bacterial species were in biofilm, both were less sensitive to Chlorhexidine digluconate and a higher concentration was needed in order to achieve the same rate of killing as for bacteria in planktonic state.
- PostAntimicrobial silk(2016) Floderus Savonen, Lotta; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringSpider silk has shown potential for use as a biomaterial. If fused to antimicrobial peptides (AMPs), recombinant spider silk could be used for medical applications and reduce the need to use conventional antibiotics to battle infections. Recombinant spider silk 4RepCT was fused to the AMPs Magainin I, Lactoferricin and a synthetically derived AMP referred to as WGR. Polystyrene disks were coated with the AMP-silk fusion proteins and the disks were incubated with cultures of Staphylococcus aureus and Escherichia coli, to test the AMP-silks antimicrobial activity. All AMP-silk fusion proteins significantly decreased bacterial adhesion of S. aureus to the disks after 48 hours of incubation compared to uncoated disks. The Mag- and WGR-silks were e↵ective already after 24 hours. The recombinant silk itself seemed to have an antimicrobial e↵ect by reducing bacterial adhesion of both bacterial strains to the polystyrene disks. Results indicated that addition of the AMPs improved this e↵ect on S. aurues, but not on E.coli.
- PostAssessment of fermentability of steam-pretreated spruce tips, needles and branches for bioethanol applications(2018) Asp, Tobias; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringThe production of bioethanol from fermentation-based bioprocesses utilizing ligno-celluosic feedstocks is an option for replacing fossil based fuels. By using genetically modified yeasts that co-consume glucose and xylose, it is possible to ferment lingo-cellulosic materials such as spruce for bioethanol production. In this project the fermentability of spruce tips, needles and branches, pretreated by acid-catalyzed steam explosion according to a design of experiments plan, was evaluated in terms of ethanol titer, rate and yield as well as cell viability. In order to ferment the spruce tips, needles and branches, suÿcient cell concentrations are needed. A preculture method was developed where enough cells were produced and harvested in the same physiological state in di˙erent batches for simultaneous saccharification and co-fermentation. A anaerobic shake flask system was used to ferment the spruce tips, needles and branches. Final ethanol titers of up to 10 g l-1, average volumetric ethanol production rates of up to 0.35 g l-1 h-1 and final yields of ethanol on available glucose and on available glucose together with xylose of up to 0.19 gEthanol gGlucose-1 and 0.16 gEthanol gGlucose+Xylose-1 respectively were observed. The final cell concentrations, colony forming units and growth rates observed were all fairly low values of up to 3.00 x 105 cells ml-1, 1.19 x 107 CFU ml-1 and 2.36 x 10-2 h-1 respectively. Even though there may be some indications on preferable pretreatment conditions in terms of fermentability, more testing and experiments are required to make statisti-cally significant recommendations.However, trends observed in this project points to either high temperature and short time or low temperature and long time as prefer-able pretreatment process conditions. Materials pretreated under these conditions showed the highest final titers and yields.
- PostAttempted Isolations of Cholesterol-to-Coprostanol Reducing Bacteria in the Human Gut(2023) Antonsson, Selma; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Landberg, Rikard; Tremaroli, ValentinaAtherosclerotic cardiovascular disease (ASCVD) is the leading cause of death globally. In early development of atherosclerosis, retention of cholesterol in artery walls is a key step. Cholesterol both endogenously produced and absorbed from the diet ends up in the gut, where bacteria may reduce it to coprostanol. Unlike cholesterol, coprostanol cannot be reabsorbed into circulation from the intestines, leading to long-standing hypotheses that high cholesterol-to-coprostanol conversion may lower blood cholesterol and thus the risk of ASCVD. Metagenomic evidence for both conversion being health-associated and ASCVD being a microbiota-modulated disease is mounting, emphasising the potential importance of microbial cholesterol metabolism in the gut. This project thus aimed to isolate cholesterol-converting bacteria from the human gut to further characterise them. Conversion was initially studied in a pure Eubacterium coprostanoligenes culture as a positive control. However, the type strain was quickly outcompeted by a contaminant whereupon culturing of faecal samples from two healthy donors was initiated. Over four sample series, several media compositions and cultivation approaches were investigated. Coprostanol was not observed in any culture. Pathway intermediates could not be analysed and as such it is possible, although improbable, that partial conversion took place. There was also no way of detecting the potential presence coprostanoligenic but non-converting bacteria. Altogether, these results reiterate on the previously established fastidiousness of coprostanoligenic bacteria. Further attempts would benefit from more research determining the conditionssupporting cholesterol conversion, as a better understanding of those conditions is a first step in characterising the bacteria performing conversion. Eventually, this might enable research on supporting their growth using prebiotics or the feasibility of their application as next-generation probiotics for improved cardiovascular health.
- PostBiofilm Reduction Modelling in a Drip-flow Reactor. Developing a Method for Growing Biofilms in a Laboratory Setting, to Evaluate the Effects of an Anti-fouling Product Used in Paper Machines.(2020) Frithiofson, Emil; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Norbeck, Joakim; W. Janco, EmmaBacterial biofilms naturally form in paper machines, causing problems in production. Reducing the amount of biofilm formed is therefore of interest to the paper industry. To model biofilm reduction, a method was developed for growing biofilms in a laboratory setting at BIM Kemi Sweden AB. The goal was for the method to be used to evaluate effect of the anti-fouling product Bimogard produced by the company, a non-biocidal agent that reduces formation of process disrupting biofilms in paper machines. Initial experiments were carried out using petri dishes for cultivation, but the main part of the work was carried out using a drip-flow biofilm reactor. The biofilms were quantified by staining with safranin and measuring absorbance. Different process parameters for running the reactor were examined and improved, including what medium concentration to use and whether to inoculate with a mono-culutre of Pseudomonas fluorescens or a co-culture where Bacillus subtilis was also added. The final method, using the mono-culture and 7.5% of standard medium concentration, was used to evaluate the effects of adding Bimogard to the nutrient medium. The addition of Bimogard significantly reduced the amount of biofilm formed, but only at low concentrations.
- PostBiophysical approaches in a structure-guided SMYD3 ligand discovery(2019) Talu, Martin Johannes; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Norbeck, Joakim; Talibov, Vladimir O.In eukaryotic cells, DNA is wrapped around nucleosomal cores formed of protein heterooctamers, which consist of core histones. Nucleosomes are the main units of chromatin organization. Chromatin exist in two states - either as eu- or heterochromatin, which either promotes or silences gene expression as a consequence of its packing states. Eukaryotic cells have developed epigenetic regulation to control the chromatin state, and to guarantee a high level of differentiation. The basis of epigenetic regulation are patterns of post-translational modifications of the nucleosomal proteins. These modifications are performed by epigenetic enzymes. This thesis focuses on one of these enzymes - the human lysine methyltransferase SMYD3. SMYD3 is also capable of interacting with certain cytosolic proteins, such as the molecular chaperone HSP90 - the human Heat Shock Protein 90. Both proteins are of high interest in the drug research and development landscape, as a drastic change in their activity and expression levels have both been shown to be related to several cancers or neurodevelopmental diseases. In this work, various truncated forms of HSP90 were produced and probed for their interactions with SMYD3 using surface plasmon resonance-based biosensor technology. A previously reported interaction of SMYD3 with the C-terminal domain of HSP90 was confirmed, with an affinity discovered to be KD = 1.3 × 10−5M. Additionally, the biosensor-based assay was used to test potential ligands of SMYD3, including low affinity fragment-like organic molecules. To complement the study, extensive crystallization and co-crystallization trials were carried out with SMYD3. As a result, conditions for the formation of various crystal forms of SMYD3 were mapped, with the best crystal form found to have high stability and good diffraction properties. A set of experiments presented herein develops expertise in the tools one can use for an efficient and rational ligand discovery campaign targeting SMYD3 histone methyltransferase.
- PostBioprospecting for novel laminarin-degrading enzymes in marine microorganisms - A step towards the use of macroalgae in bioprocesses(2015) Olsson, Joakim; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringCurrently, efforts are being made to find alternative biomasses for biofuel production, which are not competing with food production. Several kelp species have potential for large-scale cultivation, harvest and biorefinery processes. On the Swedish west coast, Laminaria digitata and Saccharina latissima are two such species. These kelps can contain up to 33% of the storage carbohydrate laminarin (dry weight), which can be hydrolysed to glucose and subsequently converted into bioethanol by for instance yeast. Enzymes able to digest the β-1,3 or β-1,6 bonds of the laminarin, to release glucose, are in large unknown and this project has been about finding microorganisms expressing such enzymes, known as laminarases. Samples from partly decomposed L. digitata and S. latissima specimens were streaked on nutrient agar plates for proliferation and isolation of surface microorganisms. Isolated organisms were subsequently screened for growth on laminarin and promising strains were identified through 16s/18s rRNA sequencing. After growth experiments on liquid medium with different carbon sources, two bacterial strains, Pseudoalteromonas ssp., were selected for further characterisation. The two strains were grown in algae extract and the sugar composition was monitored over time. In the extract, mainly mannitol and laminarin were present but glucose was formed during the cultivation, indicating the presence of hydrolytic enzymes, after which the hydrolytic activity on pure laminarin was investigated. The supernatants of one of the strains showed in vitro activity, further indicating the presence of extracellular hydrolytic enzymes. More investigations are needed on whether the enzymes can be used in any type of process. This thesis work has been successful in isolating marine organisms with laminarin-degrading activity.
- PostCartilage repair with the use of adipose tissue and 3D bioprinting(2018) attfors, madeleine; Chalmers tekniska högskola / Institutionen för kemi och kemiteknik; Chalmers University of Technology / Department of Chemistry and Chemical Engineering
- PostCharacterization of chocolate microstructure: development and comparison of microscopy techniques to evaluate cocoa butter crystallization(2013) Balestra, Lucile; Chalmers tekniska högskola / Institutionen för kemi- och bioteknik; Chalmers University of Technology / Department of Chemical and Biological Engineering
- PostCharacterization of non-homologus end joining in Mycobacterium tuberculosis using nanofluidics(2023) Persson, Elin; Chalmers tekniska högskola / Institutionen för life sciences; Chalmers University of Technology / Department of Life Sciences; Westerlund, Fredrik; Budida, Anusha; Pavlova, EvgeniyaDNA, the building block of all life, is constantly exposed to various pressures that can damage this vital molecule. These damages, if not repaired correctly, can result in mutations, disease and possibly cell death; why the cell has developed several ways to defend itself from the inevitable occurrence of DNA damages. The most dangerous form of DNA damage is double stranded breaks, for which the cell has only two repair mechanisms. One of these, non-homologous end joining, is especially important due to the fact that it repairs these dangerous breaks in situations where no template is available. However, the bacterial non-homologous end joining system lacks comprehensive understanding, especially on the level of individual molecules. Hence, the primary objective of this thesis is to explore the mechanisms of the two key players of this system. While the human system is a complex interplay of numerous proteins, the bacterial system primarily consists of two proteins that are homologous to their eukaryotic counterparts: the homodimer Ku and DNA ligase D. This project aimed to express, purify and utilize these two proteins from Mycobacterium tuberculosis to investigate their interaction with DNA in both bulk phase and with single molecule assays, especially a nanofluidic device based on confinement of DNA. Our findings reveal that the C-terminal arm of the Ku protein, as well as the nature of the double-stranded break, plays a critical role of DNA binding and bridging. The Ku from Mycobacterium tuberculosis was also found to have the ability to bridge blunt ended DNA, a characteristic not observed in other bacterial species such as Bacillus subtilis. Further, it was proven by single molecule methods that the ligation of double stranded breaks is directly dependent on efficient bridging of the DNA by the Ku homodimer.
- PostCharacterization of PlyAZ3aT(2018) Sundström, Clara; Chalmers tekniska högskola / Institutionen för biologi och bioteknik; Chalmers University of Technology / Department of Biology and Biological EngineeringAntibiotic resistance among bacteria is a growing problem all around the world. Hence, new alternatives to antibiotics are demanded for. Bacteriophages (phages) represent such an alternative, as they are viruses that naturally affect bacteria. After infecting a bacterium, the phage uses enzymes to escape the host cell and kill it. Endolysins are one group of enzymes used in this step to cut open the bacterial peptidoglycan. A future perspective on antimicrobial agents is the direct usage of endolysins to treat bacterial infections. In the lab of Grégory Resch (Lausanne University, Switzerland) a new endolysin, PlyAZ3aT, was identified within the genome of Streptococcus tigurinus AZ3aT and its activity was evaluated in a series of experiments. In this report the explorative work on PlyAZ3aT have been continued by means of comparison with the formerly known endolysin Cpl-1 and chimeric enzymes (chimeras) constructed by domain swapping between the two native enzymes (PlyAZ3aT and Cpl-1). Six chimeras were purified from crude extract using Fast Protein Liquid Chromatography (FPLC). Thereafter, the activity of all enzymes was evaluated in vitro on S. pneumoniae D39 and S. tigurinus AZ3aT using turbidity, Minimal Inhibitory Concentration (MIC) and time-kill experiments. While all enzymes showed similar activity in poor medium (NaCl 0.9%), enzymes harbouring the Cell Wall Binding Domain (CWBD) of PlyAZ3aT performed significantly better on both strains in rich medium. Whether these first interesting results reflect a negative medium effect on Cpl-1 or an activity improvement provided by the PlyAZ3aT CWBD on dividing cells remains to be determined. In addition, to further evaluate the potential of PlyAZ3aT and/or any of the chimeras as antimicrobial agent, their activity should be verified in vivo.