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Improving the efficiency of yeast by evolving them in millions of parallel pico-liter reactorsExperimental (Bachelor or Master) Position filled
Rinke van Tatenhove
Many systems show a trade-off between speed and efficiency. Think for instance of a car: when you drive fast it is less fuel efficient than when you drive slower, which means that when you drive fast you can drive less kilometers per unit of fuel. For the growth of the yeast S. cerevisiae it is the same: when this yeast grows fast it is generally less efficient in converting glucose to biomass, which means that it can make less biomass from one unit of glucose. This inefficient but fast growth in yeast usually occurs when the glucose concentration is high, while S. cerevisiae grows slow and efficient when the glucose concentration is low.
We would like to study this trade-off between growth rate and efficiency by finding efficient mutant strains regardless of the glucose concentration. Can we select for a yeast strain with a high efficiency in the presence of high glucose concentrations?
Yeast cells normally grow in suspension, sharing their environment and the available substrate. However, an efficient mutant cannot be enriched in suspension, because such a mutant will grow slower than its fast growing neighbors. Efficient but slow mutants can therefore not be selected in a shared environment. To be able to select efficient cells we separate them from each other in medium droplets floating in oil. In such a water-in-oil emulsion we have millions of separated pico-liter compartments, in which each cell has its own substrate pool and its own extracellular space. All these individual compartments can be analyzed in seconds and mutants can be picked out with a flow cytometer, allowing for high-throughput mutant selection.
In this project you will develop a platform to select efficient yeast strains, using water-in-oil emulsions. You will design a medium which is suitable to perform these selections and you will apply this method to select efficient yeast cells at a high glucose concentration.
S. pombe as an alternative model for eukaryotic central metabolismExperimental (Master)
Position filled
Johan van Heerden
The two distantly related yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, have been instrumental in our current understanding of the eukaryotic cell and its processes. Much of what we know about the cell cycle and its regulation was borne from pioneering studies with S. pombe, while S. cerevisiae has been a favourite workhorse in studies on eukaryotic metabolism (especially central carbon metabolism) and textbooks on this subject are therefore filled with insights derived from this organism.

While the cell-cycle field has embraced S. cerevisiae as a complementary eukaryotic model, researchers with a focus on metabolism have yet to harvest the potential insights to be gained from systematic (and comparative) studies of S. pombe metabolism. Today, the majority of research on S. pombe is still dedicated to unraveling cell cycle regulation and other cellular functions such as DNA repair, maintenance and aging, with very little fundamental research on the central metabolism of this yeast.

While there are many similarities between these two species, there are also several important differences including: cell morphology, the mode of cell division, the position of cell-size checkpoints during cell-cycle progression, the role of glucose sensors, the ability to respire ethanol, the presence of a glyoxylate cycle and differences in mitochondrial functions.

As many of the differences between these two species pertain to metabolic functions, a better understanding of the metabolic profile of S. pombe should serve to greatly expand our compendium of knowledge on eukaryotic metabolism, beyond that of S. cerevisiae and cancer cell lines.

The project
In this project you will lay down the foundation for metabolic studies that use S. pombe as an alternative to S. cerevisiae. You will perform both physiological and metabolic characterisations of S. pombe - in experimental settings that are typical for S. cerevisiae - in order to gain detailed descriptions of (1) growth kinetics, (2) cell size distributions and (3) profiles of intracellular pH and glycolytic intermediates, under different growth conditions. It is hoped that these results will serve to identify key differences, but also similarities, between these two species and that these insights will form a basis for future research.

Batch and/or chemostat cultivations
Genetic transformations to express fluorescent reporter proteins
Enzymatic assays to determine intracellular metabolite concentrations
And, depending on the progress of the project, one or several of the following analytical techniques: HPLC, Coulter counter, Flow cytometry and Fluorescence spectroscopy, Fluorescent microscopy.

Duration of project: 5-9 months

Your CV: Ideally you will have an experimental background, with experience working in a microbiological setting.

Start date: February 2017 or later.
The regulation of glycolytic-to-gluconeogenic transitions in different yeast speciesTheoretical/Experimental (Master)
Position filled
Phillipp Schmidt
Johan van Heerden
The preferred mode of metabolism for most yeast species is glycolysis. This metabolic pathway breaks down glucose or other sugars and produces pyruvate, which can further be utilized in fermentation or respiration in order to produce energy and precursors for biosynthesis. When encountering excess levels of glucose, the baker’s yeast Saccharomyces cerevisiae performs aerobic fermentation, which leads to the formation of ethanol. This is also called the Crabtree effect. Upon depletion of glucose it can utilize the previously produced ethanol as carbon source. This metabolic transition is called the diauxic shift. In order to grow on ethanol, the cells need to synthesize glucose and other metabolic intermediates. This achieved through the gluconeogenic pathway, which is a essentially glycolysis operating in the reverse direction. Due to the irreversibility of two glycolytic reactions, new enzymes need to be expressed for this pathway including FBPase (Fructose 1,6-bisphosphatase). The regulation of this enzyme is not completely understood, even though it has been intensely studied in S. cerevisiae. For example, an open question remains regarding the specific signals that lead to induction of this gluconeogenic enzyme - are low glucose levels sufficient to signal activation of gluconeogenic enzymes, or does extracellular ethanol play a role?

To find answers to these questions, other yeast species may provide clues. For example, Schizosaccharomyces pombe, like S. cerevisiae, exhibits the crabtree effect, but cannot consume the ethanol it produces via aerobic fermentation. Does ethanol still play a role in inducing gluconeogenesis? There are also crabtree negative yeasts, like Kluyveromyces lactis, that do not produce ethanol from glucose in the presence of oxygen. Clearly, in this case ethanol cannot play a role in signaling glycolytic-to-gluconeogenic transitions.

In this project, we want to compare the dynamic regulation of glycolytic-to-gluconeogenic transitions in these three different yeast species. We will use a technique called RNA FISH to count single mRNA molecules for PFK (Phosphofructokinase, a glycolytic gene) and FBPase (a gluconeogenic gene) in individual cells as they transition from excess to no glucose. By comparing differences in the induction of FBPase between these yeast species, the aim is to gain a better understanding of the roles that glucose and ethanol concentrations respectively play in inducing gluconeogenesis.

Duration of project: 5-9 months

Your CV: Ideally you will have a strong experimental background, and some basic programming skills (e.g. Python, R, Matlab).

Start date: May 2017 or later.
Drug targeting in metabolism: from antiparasitic target to drug screening assaysExperimental (Master)
Position filled
Jurgen Haanstra
j.r.haanstra[at] and Marijke Wagner
Trypanosoma brucei is the causative agent of deadly African Sleeping Sickness. In its lifestage inside the mammalian host this unicellular parasite relies almost exclusively on glycolysis to generate ATP. As 50% inhibition of glycolysis is sufficient to kill the parasite, glycolysis is a prominent target pathway for antiparasitic drugs. A network-based analysis has shown that the glucose transporter has the highest control over the glycolytic flux in T. brucei and is therefore a prominent drug target.
To get to a semi- to high throughput assay to test drugs against the T. brucei transporter we have complemented a yeast glucose-transporter null mutant with the trypanosome transporter. In this project you will design and test drug screening assays on live T. brucei and for the yeast complementation mutant.
If desired, this project can incorporate a modelling part as we have a kinetic model of T. brucei glycolysis (and yeast).
To what extent does Lactococcus lactis need to adapt its proteome upon environmental transition?Experimental (Master)
Position filled
Sieze Douwenga
WUR supervisor: Berdien van Olst
Microorganisms like bacteria optimize their growth by adapting to their environment. When the environment changes, they might need different proteins or metabolic states to continue growing. This can result in temporary growth arrest, a lag-phase. For example, a lag phase is observed when the bacterium Lactococcus lactis is transitioned from glucose to maltose. A potential determinant of the lag phase duration is the time required to synthesize the alternate proteins required for growing in the new environment.
We do not always observe a lag phase upon transition, as L. lactis may be a generalist in certain environments. Generalists can grow under multiple conditions; and must express extra proteins which are non-essential for the current environment. This burden likely results in a reduced growth rate. It is therefore likely that conditions exist in which L. lactis specializes -instead of generalizes- in order to increase the growth rate. However, when a specialist encounters a new environment, it must synthesize the newly required proteins to continue growing. The amount of new proteins required is likely to depend on the pathway similarities between the new and old environment. On the contrary, a generalist might already have (part of) these proteins and is able to grow directly (or has a shorter lag phase). The degree of adaptation necessary to continue growing in a new environment can be determined by analyzing the complete proteome of L. lactis under various growth conditions. Additionally, characterizing the RNA, product and substrate fluxes is required, as certain proteins may be present below the detection limit.

The project
You will design and run several controlled batch reactor experiments under various conditions. From these reactors you will sample and analyze the proteome, RNA, and product and substrate fluxes of L. lactis. The project is a collaboration between the Systems Bioinformatics group at Vrije Universiteit Amsterdam (VU) and the Laboratory of Biochemistry at Wageningen University (WUR). Therefore, you will have the help of two daily supervisors (one at each university) and work at both of these sites. At the VU, you will have the facilities for running reactor experiments and for determining the product and substrate fluxes. At the WUR, you will characterize the proteome and RNA profile of L. lactis. By the end of your project, you will have obtained new insights on the proteome differences in L.lactis between the tested conditions.

• Background in molecular biology/biochemistry and previous experience in microbiological culturing
• Interest in proteins/proteomics is important
• 6-9 month availability
Variability of glycogen metabolic abilities amongst Lactobacillus crispatus vaginal isolates.Experimental (Bachelor or Master)Position filledRosanne Hertzberger (topic expertise) and Jurgen Haanstra (lab/daily support) - contact: rosanne dot hertzberger at gmail dot com
Note: This project will follow “Open Kitchen Science” principles, which means that we will aim to publish all outcomes, including methods, data, posters, positive and negative results, on a blog and on various other open science platforms (Zenodo, FigShare etc). This does not preclude publishing at a later stage in most traditional academic literature, such as the ASM journals.

Background :
The bacterial communities colonizing the vagina of reproductive-age women have a remarkable low level of diversity. In most cohorts studied sofar, a majority of women harbor bacterial communities dominated by lactobacilli. The most frequently encountered species of lactobacilli are Lactobacillus crispatus and iners. These lactobacilli produce high levels of lactate (up to 100 mM) and acidify the vagina (pH<4). The absence of Lactobacillus species is frequently accompanied by an overgrowth of various gram-negative and –variable species, commonly referred to as Bacterial Vaginosis (BV). This common vaginal microbial state is often asymptomatic, but in some cases leads to abnormal odor and secretions and is generally associated with a higher risk of acquiring sexually transmitted infection and premature birth.

Little is known about the carbon sources that fuel colonization and acidification by vaginal lactobacilli. One of the possible vaginal carbohydrates available to microbes is glycogen, which is present in high levels in the epithelial layers of the uterus, exocervix and vagina of reproductive-age women. Previous experiments suggest that various BV-associated microbes (Gardnerella and Prevotella) as well as Lactobacillus iners and some Lactobacillus crispatus isolates can utilize glycogen as a carbon and energy source.

Recently we found remarkable variability in glycogen metabolism amongst a group of ~20 vaginal isolates of Lactobacillus crispatus. This ability to use external glycogen for growth and acidification was found to correspond to a type 1 pullulanase gene variant. The strains that are able to grow using glycogen as their sole carbon and energy source have an intact N-terminal signal peptide in their type 1 pullulanase gene.
We hypothesize that this signal peptide may be involved in allocating the enzyme to the outermost S-layer which is probably important to access and breakdown the large molecules of extracellular glycogen in smaller units that can be transported over the cell membrane. The subset of Lactobacillus crispatus strains that are unable to grow using glycogen as a carbon source have various mutations disrupting the signal peptide in this area of the type 1 pullulanase gene.

Lastly, expression of the activity was only detected during growth of L. crispatus on glycogen and not on glucose. This indicates either (1) carbon catabolite repression: repression of the expression of some or all genes involved in glycogen metabolism when extracellular glucose is detected or (2) induction of genes involved in glycogen metabolism in the presence of glycogen or one of the breakdown product or (3) regulation on enzymatic level (feed-back inhibition?).

Understanding these basic aspects of vaginal microbial metabolism may lead to developing better treatment for these very common vaginal symptoms, thereby increasing reproductive health.
Hypothesis: An intact S-layer signal peptide of the Lactobacillus crispatus type 1 pullulanase is essential for allocating the pullulanase that is responsible for catalysis of the first step of glycogen metabolism to the outermost membrane layer.

Experimental plan
Currently, the evidence supporting the hypothesis consists of a link between glycogen breakdown phenotype and type 1 pullulanase genotype in a set of ~20 Lactobacillus crispatus strains (genotype-fenotype). In this project we aim to gather further evidence for the hypothesis by:
-identification of the pullulanase with various substrates (glycogen, starch, amylose) and studying production of breakdown products (maltotriose, maltopentaose, maltose, glucose) using TLC. (The enzymatic activity has been detected sofar only with a basic starch-iodine assay.)
-studying cellular location of the activity by comparing supernatants and pellets before and after disruption of the cells.
-studying the regulation of expression: tracking enzymatic activity during growth experiments on glycogen and/or glucose in various concentrations and in growth experiments perturbed with glucose, glycogen or glycogen breakdown products.
-Several basic microbial skills will be developed: anaerobic culture, enzymatic assay, medium preparation, sterile working, optical density measurements, inoculation and plating.
Analyzing the interactions between yeast and bacteria.Experimental (Bachelor). Position filledRinke van Tatenhove
In most controlled microbiological experiments the behavior of one single organisms is studied. However, in nature micro-organisms are never alone, but they live together with many different species and they interact with each other. This project focusses on interactions between yeast and bacteria. We will use a synthetic consortium in which yeast and bacteria grow together and one organism grows on a metabolic product of the other. This interaction will eventually be used to select for increased metabolite yields.
In this project you will develop a medium to co-culture yeast and bacteria and you will analyze the interactions between these organisms using a flow cytometer. You will furthermore investigate how we can attach these cells to each other, to reduce the distance between them and make their interactions more efficient.
Biodegradation of novel pharmaceutical products. Experimental (Master)Baptiste Poursat
Organic pollutants, such as pharmaceutical and industrial products, are frequently detected in the environment, especially in aquatic habitats and in wastewater treatment plant (WWTP) discharges. They are considered as “emerging contaminants” since most of them are not currently covered by existing water-quality regulations, and their effects on the environment are still poorly understood. Knowledge on environmental persistency is essential to make an efficient environmental risk assessment of new manufactured chemicals. Indeed, a key component of both hazard determination and environmental risk assessment is the accurate estimation of the degradation of a chemical in the environment. Ready biodegradation tests (RBT) from the OECD guideline are required by the European REACH regulation to evaluate the biodegradability of chemical substances produced by industry. However, RBT suffer from several drawbacks related to the heterogeneity of their inoculum, due to their different origin and history. Indeed, the source, concentration and pretreatment of the inoculum are one of the most important factors that influence the RBT results and the extent of biodegradation of chemicals in the environment. The aim of this master thesis project is to compare the biodegradation rates of several pharmaceutical and industrial products (using LC-MS/MS) by different inocula from municipal, hospital and industrial WWTPs. Microbial communities will be characterized by Illumina sequencing and metagenomic analyses. Laboratory work for this project will mostly be performed in the laboratory of the University of Amsterdam (Science Park). For more information, please contact Baptiste Poursat, Dr John Parsons (UvA-ESS) or Dr Rob Van Spanning (VU Amsterdam).
The role of metabolic interactions in antibiotic toleranceTheoretical (Master)Bas Teusink
Bacteria in polymicrobial infections interact with each other. These interactions can change the environment to such an extent that some bacteria can become more or less susceptible to antibiotics. Metabolic interactions likely underlie this antibiotic tolerance effect. By means of metabolic modeling you will investigate some of these potential antibiotic-tolerance interactions in polymicrobial infections.

This internship is a cooperation with Dr Marjon de Vos from Wageningen University

Bacteria in polymicrobial infections interact with each other. These interactions can change the environment to such an extent that some bacteria can become more or less susceptible to antibiotics. Metabolic interactions likely underlie this antibiotic tolerance effect. By means of metabolic modeling you will investigate some of these potential antibiotic-tolerance interactions in polymicrobial infections.

This internship is a cooperation with Dr Marjon de Vos from Wageningen University
Segmentation of microscopy images using deep learningTheoretical (Bachelor or Master)Philipp Savakis
Under dynamic conditions (changes in nutrient availability, ion concentrations, toxic compounds etc.), individual cells within a population show different responses. In order to quantify these responses, we aim to use a combination of microfluidics, phase contrast, and fluorescence microscopy. Images taken with the microscope need to be pre-processed for further analysis: the analysis algorithm needs to know in which part of the image the cells are located and what their shape is. So, in the first part of the data analysis, we need to solve a pixel-wise classification problem. The project focuses on implementing deep learning techniques (convolutional neural networks in caffe) to generate segmented images, which are then fed into a downstream processing pipeline. Proficiency in python and a general interest in deep learning techniques and pattern recognition are required, knowledge of Matlab/octave and caffe would be very helpful.