Global ETD Search

41	Toward prototyping metabolic pathways in cyanobacteria using cell extracts Bensabra, Amina January 2022 (has links) Cyanobakterier är intressanta mikroorganismer för produktion av biobränslen från solljus, vatten och atmosfärisk koldioxid och anses därför vara potentiella mikrobiella cellfabriker. Men på grund av långsam tillväxt och låg produktion är genteknologi processen intensiv och tidskrävande för cyanobakterier. En alternativ metod till prototypteknik för metabola vägar är att använda cellfri metabolisk teknik där cellysat av överuttryckta enzymer används. I detta projekt försökte vi utveckla en metod för cellfri metabolisk ingenjörsteknik för cyanobakterien Synechocystis PCC 6803 med hjälp av den övre mevalonatvägen som exempelreaktionsväg. Vi började med att utveckla tre fluorescensbaserade metoder för att detektera proteinöveruttryck med hjälp av de tre enzymerna från mevalonatreaktionsvägen. Dessa metoder använde fusering av YFP-proteinet till målproteinet, en delad GFP-reporterprotein eller translationskoppling. Ett av de överuttryckta enzymerna verkade vara giftigt för Synechocystis-celler så flera inducerbara promotorer användes för att försöka uttrycka enzymet. Den högst uttryckande konstruktionen för varje gen valdes ut och proteiner extraherades och blandades i en cellfri metabolisk ingenjörsreaktion. Även om inget mevalonat kunde detekteras med hjälp av gaskromatografi i detta projekt, berodde detta sannolikt på otillräckligt högt proteinöveruttryck av mevalonatgenerna. / Cyanobacteria are desirable microorganisms for the production of biofuels from sunlight, water and atmospheric carbon dioxide, and are therefore considered potential microbial cell factories. But due to slow growth rate and low production rates, the engineering processes for bioproduction is labour intensive and time consuming. An alternative method to prototype metabolic pathway engineering is to use cell-free metabolic engineering, where cell lysates of enriched enzymes are used. In this project, we attempted to develop a method for cell-free metabolic engineering for the cyanobacterium Synechocystis PCC 6803 using the upper mevalonate pathway as an example pathway. We started by developing three fluorescence-based methods for detecting protein overexpression using the three enzymes from the mevalonate pathway. These methods used YFP fusion to target proteins, a split GFP reporter tag or translation coupling. One of the overexpressed enzymes appeared to be toxic to Synechocystis cells so several inducible promoters were used to try and express the enzyme. The highest expressing construct for each gene was selected and proteins were extracted and mixed in a cell free metabolic engineering reaction. Although no mevalonate could be detected using gas chromatography in this project, this was likely due to insufficiently high protein overexpression of the mevalonate pathway genes. cyanobacteria metabolic engineering cell-free metabolic engineering enzymes sustainability cyanobakterier metabolisk genteknik syntetisk biologi enzymer hållbar bioproduktion Biochemistry and Molecular Biology Biokemi och molekylärbiologi
42	Computational and experimental approaches to regulatory genetic variation Andersen, Malin January 2007 (has links) Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations. The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease. An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available. The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene. Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect. Molecular biology Genetics single nucleotide polymprhism (SNP) regulatory SNP transcription factor binding site phylogenetic footprinting allelic imbalance EMSA CD36 cardiovascular disease. Genteknik inkl. funktionsgenomik
43	Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea Elison Kalman, Grim January 2019 (has links) This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR. structural biology cell culture protein expression genetic engineering spectroscopy X-ray crystallography small angle X-ray scattering SAXS transcription factor PerR metal binding antibiotics Saccharopolyspora erythraea S. erythraea soil bacterium actinobacteria actinomycetes oxidative stress peroxide stress structure determination characterization stabilization peroxide sensor iron sensor manganese sensor metalloprotein metal ion homeostasis metal ion prokaryotic strukturbiologi cellodling proteinuttryck genteknik spektroskopi röntgenkristallografi småvinkel-röntgenspridning SAXS transkriptionsfaktor PerR metallbindande antibiotika Saccharopolyspora erythraea S. erythraea jordartsbakterie Aktinobakterier Actinobacteria Actinomycetes oxidativ stress peroxid stress strukturbestämning karaktärisering stabilisering peroxidsensor järnsensor mangansensor metalljonshomeostas metalljoner prokaryot Structural Biology Strukturbiologi

Page generated in 0.0544 seconds