SCK.CEN (Studiecentrum voor kernenergie)
Voorstel stage-onderwerp 2012-2013: Structural prediction of CopQ family proteins
The soil bacterium Cupriavidus metallidurans CH34 is tolerant to a wide range of toxic metals.
Whole-genome DNA microarrays demonstrated in this organism common themes between metal and oxidative stress defence mechanisms. A small number of conserved hypothetical genes (i.e. genes whose product have a match with protein databases but for which no function is known) were highly expressed under both stress conditions. These genes, some of which were linked to metal resistance loci, encode short proteins with some striking features (coined the CopQ family). They all possess a distinctive signal peptide, and thus are secreted to either the periplasm or the exterior, and share multiple repetitive motifs that are evenly spaced. This repeat motif always centers around a tyrosine or phenylalanine. Because aromatic ring structures in proteins are particularly sensitive to reactive oxygen species (ROS), these Tyr- and Phe- containing motifs may play a special role in metalinduced redox reactions or ROS scavenging.
A first BLAST analysis indicated that orthologous sequences were only found within the
proteomes of Cupriavidus and Ralstonia species. In addition, a first structure prediction with the
PHYRE server indicates that CopQ-family proteins may form a single linear helix in which
aromatic Tyr and Phe residues are exposed at the outside (hence they would be available for
ROS interaction). These preliminary findings need to be consolidated.
The apprentice will use an extended motif search using the biogrep algorithm applied on very large protein sequence datasets to confirm that CopQ-family proteins only occur in the genera Ralstonia and Cupriavidus subclassification of the CopQ-family will be performed by multiple sequence alignment and domain analysis selected CopQ-family proteins will be subjected to a thorough structural prediction using a variety of softwares and public webservers (to be identified and tested by the apprentice).
Voorstel stage-onderwerp 2011-2012
Arthrospira are photoautotrophic filamentous cyanobacteria that typically reside in soda lakes. They have a high protein content, are rich in carbohydrates, essential fatty acids, and a variety of minerals, vitamins, and nutritional pigments such as b-carotene. Arthrospira species have a long history of human consumption (being commercialised under the name ‘Spirulina’) and are used worldwide as feed for fish, poultry, and farm animals. Besides their nutritional value there is now also a growing interest in Arthrospira species for their clinical relevance (e.g. as antioxidants), potential in bioremediation and biosequestration, and possible applications in biofuel technology.
Arthrospira sp. PCC 8005 was selected by the European Space Agency as an oxygen producer as well as a nutritional endproduct for the life support system MELiSSA. Whole-genome shotgun sequencing of strain PCC 8005 resulted in a 6,3 Mb draft sequence with 5,856 protein-coding reading frames (Janssen et al., Journal of Bacteriology, May 2010, p. 2465-2466, Vol. 192, No. 9). Other genome draft sequences currently available are those of Arthrospira platensis strain NIES-39, A. platensis strain Paraca, and the related species A. maxima CS-328.
making an inventory of all Arthrospira genes related to carbon fixation, carbonate transport, carotenoid synthesis, photosynthesis (including light-harvesting complexes, photosystems I and II, thylakoid structures, cytochromes, and electron transport mechanisms), membrane bioenergetics, nitrogen metabolism, respiration, glycogen synthesis, and fermentation. This will entail comparative analysis based on all-to-all BLAST operations, the use of the GenBank and CyanoClust databases and the MaGe platform (http://www.cns.fr/agc/microscope/).
The apprentice may also focus on certain loci, preferably in the frame of ongoing experimental research (e.g. genes involved in oxidative stress or DNA repair, R-M systems, CRISPR elements, and phylogenetic studies of psbA paralogs and -orthologs encoding different D1 protein isoforms).
Abstract 2018-2019 (1) advanced bachelor of bioinformatics: METAGENOMICS TOOLS BENCHMARKING AND OUTPUT UNIFICATION
The ever-growing demand for data is bigger than ever. In a continuously evolving world of computing, methods have to be developed to make this big amount of data interpretable and understandable. In the field of microbiology, metagenomics is becoming a key player in determining bacterial, archaeal and even eukaryotic species present in vast and complex microbiomes. Culturing microbiome strains has proven to be challenging, as has the process of determining species from shotgun sequencing approaches. After sequencing, reads are assembled into contigs, which then serve as basis for scaffolds, which can finally be used to close genomes. The clustering of contigs (also known as binning) is a crucial step in correctly assembling a genome from metagenomics samples. The performed research, attempts to identify suitable binning programs based on the binning output generated. The selection of these programs is based on a comprehensive literature study, identifying CONCOCT, MaxBin2.0 and MetaBAT2 to be most suitable and user friendly for the tested sample of interest. A python script was developed to support analysis of the output of metagenomics tools. This script generates a summary per selected binning program, yielding information about its overall performance, as well as provide summary statistics for the sample of interest.
Abstract 2018-2019 (2) advanced bachelor of bioinformatics: POST-PROCESSING ANALYSIS AND VISUALISATION FOR THE 16S rRNA METAGENETICS DATA
By detecting marker genes like the 16S rRNA in bacteria, an overview of the community and its relations between species can be studied using high-throughput sequencing technologies. To grasp a better understanding of 16S rRNA metagenetics, several pipelines (like mothur, QIIME, OCToPUS or LotuS) have been designed to process these sequencing results and allows us to investigate these microbial communities. An in-house pipeline has been developed, within SCK●CEN, that performs post-processing analysis of the output of these pipelines. Yet there was a need to visualize these results and present it for the users in a comprehensive report. Here we present a visualization and analysis pipeline developed in R. This tool takes the output of the SCK●CEN in-house pipeline, creates a report describing the alpha and beta diversity, performs biomarker detection and presents hypothesis testing based on the meta information of the experiment. As a demonstration, a mouse experiment was processed using the OCToPUS pipeline, followed by an analysis and visualization using the post-processing tool. The tool will be later integrated with the OCToPUS pipeline into one comprehensive package for assessing any 16S metagenetics data.
Samenvatting eindwerk 2014-2015: Optimatization of long-term storage procedures for the prebiotic cyanobacterium Arthrospira sp.
Arthrospira sp. is a well know prebiotic cyanobacterium due to its high protein concentration and is used as “superfood” in the form of food supplements under the name Spirulina. Thanks to this characteristic and its ability to produce oxygen by means of photosynthesis, Arthrospira sp. can be used as oxygen and food source. Therefore it is an excellent choice for the waste recycling system for long-term space missions (MELiSSA-project). Nowadays Arthrospira sp. is kept alive via sub-culturing. However this is not an optimal way of conservation due the high risk of contamination, possible genetic drift and human work load. A storage procedure for storing Arthrospira sp. in an dormant but reversal way is still to be discovered. For that purpose, diverse preservation methods were tested: Preservation in agar under different storage conditions; preservation using cryoprotectants like DMSO and trehalose; and preservation following metabolic conditioning such as nitrogen starvation and natural trehalose accumulation following salt stress. After each test, the cell integrity and cultivability were checked by microscopic imaging, including fluorescence microscopy, flow cytometry and sub-culturing. The obtained results showed that preservation using 10% DMSO and 5% trehalose and 10% trehalose is effective for one week preservation, also preservation in agar shows positive results after one week preservation. Concerning metabolic conditioning, trehalose accumulation was successfully measured but, these experiment is still running so final results are still to come. Nitrogen starvation showed encouraging results while the use of RNA later was not convincing.
Samenvatting eindwerk 2013-2014: Optimization of preservation procedures for Arthrospira sp. in the frame of the MELiSSA (Micro-Ecological Life Support System Alternative) project
Probiotic cyanobacteria, such as Arthrospira sp., are daily used as nutritional supplements. They are considered for promising applications beyond Earth, where they can play a crucial role in closed miniaturized biological waste recycling systems, like the MELiSSA project, that is currently developed to support future long-term space missions. Cyanobacteria can be used to remove CO2 from the space cabin, to produce oxygen, to remove nitrogen compounds from waste water and to produce edible biomass. Today, Arthrospira strains are maintained in continuous culture, and new batches are inoculated from previous batches. It is presently unknown how to store Arthrospira sp. in an inactive but reversal state, even for short period of time. Because the several years length of a mission to Mars, particular strain conservation and inoculation strategy to start new cultures have to be developed for space applications. In this study cell integrity and cultivability after various preservation procedures have been assessed by optical density- and pH-measurements, microscopy and flow cytometry. These procedures included preservation in Zarrouk agar medium, alginate and in presence of clay, all in different storage conditions and over different periods of time. Also, drying on filters, lyophilization and stepwise freezing with cryoprotectants were assessed. In the results obtained from these experiments, it was observed that preservation in presence of clay was the most successful as an alternative to preservation in Zarrouk liquid medium. Also preservation in agar could be promising on the condition that a lower concentration of agar is used. Cryopreservation with a mixture of DMSO and trehalose and drying in presence of clay after induced trehalose accumulation also showed to be promising applications.
Samenvatting eindwerk 1 2011-2012: Ontwikkeling van Illumina data analyse pipeline voor resequencing
Aan SCK-CEN (studiecentrum voor kernenergie), afdeling microbiologie werkt men mee aan de MELGEN projecten (Melissa Genetic Stability study), die deel uitmaken van het MELiSSA ecosysteem onderzoek (Micro-Ecological Life Support System Alternative). Hierin bestudeert men onder andere de Rhodospirillum Rubrum bacterie. Het huidige doel is, het onderzoeken van de genomic verschillen tussen de Rhodospirillum Rubrum stam S1 ten opzichte van de S1H stam.
In deze context was er nood aan een uniforme pipeline die de trage, niet-objectieve manuele zoektocht naar variaties in resequencing data kon vervangen en verbeteren. Door de constante daling van de sequenerings prijs per genoom is de vraag naar een dergelijke tool nog sterker toegenomen.
Door optimaal gebruik te maken van de paired-end read technologie kunnen we voorspellingen doen over deleties en inserties in de mutant. Door gebruik te maken van SAMtools kunnen we ook SNP’s (single-nucleotide polymorphism) identificeren.
Wij hebben een robuuste pipeline gebouwd die deleties en inserties groter dan 200bp kan detecteren met een aanvaardbare kwaliteit. De pipeline is nog niet volledig gevalideerd, maar de eerste resultaten zijn positief.
Samenvatting eindwerk 2 2011-2012: Vergelijkende studie van Arthrospira genomen
De cyanobacterie Arthrospira werd door de European Space Agency geselecteerd voor de productie van zuurstof en als eetbaar eindproduct in het gesloten levensondersteunend systeem MELiSSA. Het is de bedoeling dat dit systeem, dat voorziet in de volledige herwinning van menselijk afval (faeces, urine, plantenresten, papier, etc.), kan worden gebruikt voor verre ruimtereizen of voor een permanente ruimtebasis. De keuze voor Arthrospira is niet zo vreemd omdat deze bacterie fotosynthetisch is, dus haar energie haalt uit zonlicht, CO2 benut als koolstofbron, en water kan oxideren tot zuurstof. Bovendien werd deze bacterie reeds in de oudheid gebruikt als voedingssupplement en is Arthrospira vandaag een belangrijk commercieel product, met een geschatte wereldproductie van 50,000 ton per jaar.
De onderzoekseenheid Microbiologie van het SCK•CEN onderzoekt de genetische stabiliteit van de bacteriën die gebruikt worden in de MELiSSA reactor. Daarom werd in 2009 besloten om het volledige genoom van de Arthrospira stam PCC 8005 te sequeneren. Deze stam is sinds 1994 in gebruik door de European Space Agency. In de afgelopen twee jaar werden door andere onderzoeksgroepen, gesitueerd in de Verenigde Staten, Japan, en Thailand, de genomen van drie andere Arthrospira species of stammen gesequeneerd. Deze data zijn vrij beschikbaar via publieke databanken. Omdat de vier genoomprojecten dezelfde problemen ondervinden ten gevolge van het hoge aantal repetitieve sequenties werd getracht de vier genomen met elkaar te vergelijken. Dit bleek maar tot op zekere hoogte mogelijk mede door de hoge repetitiviteit maar ook wegens de veelvuldige DNA herschikkingen in de vier genomen.
Deze studie bestond uit drie delen: vergelijkende genoom analyse op DNA niveau, sequentie similariteitsonderzoek op eiwit niveau, en een fylogenetische analyse van twee enzymes van de citroenzuurcyclus.
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