Dirk is currently a PhD student with Prof Andrew deMello in the Institute for Chemical and Bioengineering at ETH Zurich. He has an MEng in Bioengineering from Imperial College London, with the first two years spent taking engineering and medical science courses in the Bioengineering Department, and the third year in the Electrical Engineering Department. His final undergraduate year was spent abroad at University of California, Berkeley, where he worked with Prof Luke Lee in the Bioengineering Department and took classes in BioMEMS, microfluidics, robotics, and molecular biomechanics.
Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into in the dynamic interactions between the basidiomycete Coprinopsis cinerea and Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of Bacillus subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.
A continuous flow method for the suspension of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids in oil using a microfluidic platform is presented. The system consists of a microfluidic device housing a semipermeable membrane, a vacuum pump, and a syringe pump. Separation is achieved using a counter current flow of chloroform and a lipid containing oil stream, driven by the syringe pump and vacuum. Using such a system, a high efficiency extraction method was realized through the use of a semipermeable polydimethylsiloxane (PDMS) membrane on an anodized aluminum oxide (AAO) support. For a liquid flow rate of 5 μL/min, an air flow rate of 100 mL/min, and initial chloroform concentrations between 0.245 and 1.619 M, extraction rates of 93.5% to 97.9% and a retentate stream purity of between 99.79% and 99.29% were achieved.
We present a novel connector that allows for easy handling and injection of sample volumes between 1 and 20 μl. All tubing connections between external pumps and the microfluidic device are established before the sample is introduced into a sealable reservoir built into the connector. This approach allows for multiple injections of small sample volumes without the need to dismantle the chip-tubing assembly. We demonstrate that the connector reservoir seal can withstand pressures of up to 6 bar, that opening or closing the reservoir does not dislocate the sample by more than 35 nl, and that the connector can be used for injecting samples into both miscible and immiscible carrier fluids.
Liposome structures have a wide range of applications in biology, biochemistry, and biophysics. As a result, several methods for forming liposomes have been developed. This review provides a critical comparison of existing microfluidic technologies for forming liposomes and, when applicable, a comparison with their analogous macroscale counterparts. The properties of the generated liposomes, including size, size distribution, lamellarity, membrane composition, and encapsulation efficiency, form the basis for comparison. We hope that this critique will allow the reader to make an informed decision as to which method should be used for a given biological application.
The habitat of the model basidiomycete Coprinopsis cinerea is the nutrient-rich dung of herbivorous animals which is also populated by various bacteria. Bacteria and fungi share a lifestyle of nutrition by absorption. Due to trophic competition strategies to defend the niche have evolved in both organisms. However, these antagonistic strategies are not well characterized on a cellular level. To visualize morphological changes of fungal hyphae in co-cultivation experiments at a cellular level is technically challenging due to the three-dimensional growth pattern of a fungal mycelium. To get insight into these interactions new microfluidic platforms were developed. These platforms allow fungal hyphae to grow into microchannels that restrict fungal growth in one dimension and thus reduce the complexity of the fungal mycelium. Subsequently, bacteria can be co-inoculated. This novel technology allows one to observe the same hyphal compartment in a confined environment and the behavior of a bacterial population over time. This novel approach was used to investigate the confrontation of C. cinerea with Bacillus subtilis. Bacillus is a species known to exhibit antifungal activity and antibacterial peptides active against Bacillus secreted by C. cinerea were identified recently. In the presence of B. subtilis the hyphae stopped growing after five hours of co-cultivation. The growth stop coincided with the presence of collapsed hyphal compartments and concomitant emergence of membrane-enclosed cytoplasm. Upon confrontation with a B. subtilis mutant that does not produce the known antifungal lipopeptide, fengycin, C. cinerea hyphae did not show collapsed hyphal compartments but were still inhibited in growth. Finally, it was observed that B. subtilis cells attached in an end-on manner to only a subset of C. cinerea hyphae indicating hyphal differentiation within the vegetative mycelium. The difference between these two subsets of hyphae will be further analyzed. The novel experimental set-up opens new perspectives and insights on fungal interaction studies.
In the soil biosphere, fungi are confronted with many different antagonists and to combat these, they evolved an impressive arsenal of toxic defense molecules. Their production is often developmentally regulated but can be induced upon confrontation with the antagonist. The regulation, spatial distribution and specificity of this inducible defense response is however poorly understood. To address these questions, we study the interaction between Coprinopsis cinerea and the fungivorous nematode Aphelenchus avenae. C. cinerea responds to nematode insult by transcriptional induction of nematotoxic lectins in the vegetative mycelium. To investigate the dynamics of the interaction, we created a C. cinerea reporter strain, in which the induction of a toxic effector lectin can be followed by the expression of the dTomato protein. Additionally, we designed microfluidic devices, which allow monitoring of the defense response in individual hyphae, in real time. Using this approach, we showed that the lectin-mediated defense of C. cinerea is specific to A. avenae and is not triggered by other stimuli, such as bacteria or hyphal damage. By restricting the movement of A. avenae, we could further show that C. cinerea elicits this defense response only locally and not systemically within the mycelium. However, our data indicate that the mycelium of C. cinerea contains different hyphal subtypes of which one is able to transmit this induction over large distances. Finally, the confinement of A. avenae within the microfluidic device allowed the extraction of only highly induced mycelial parts for quantitative gene expression analysis. In comparison to conventional methods, which involved analysis of complete mycelial colonies, we show that the level of upregulation for defense genes was drastically underestimated.
Plant roots are highly sensitive to changing environmental conditions such as water and nutrient availability, biotic and abiotic stresses. Long-distance communication from root to shoot has recently been shown to be specific for salt stress perceived by roots (Choi et al., 2014). Open questions include how and why certain stimuli cause local, cell-autonomous responses, whilst others result in cell-cell communication and coordinated responses by tissues, organs or the entire plant. To understand how roots sense and process the information about their environment we need tools that allow live imaging of roots and provide precise control over the root microenvironment. Over the past years, a number of microfluidic devices have been developed to cultivate and perfuse Arabidopsis roots at the microscope. These devices have substantially advanced experimental access to roots and some devices, e.g. the RootChip, allowed pulsed treatments (Grossmann et al., 2011). So far, it has not been possible to locally apply treatments only to selected regions of the root, as the organ was generally perfused as a whole. Moreover, due to roots bending within observation chambers, an even perfusion was often challenging to achieve, thus affecting reproducibility of the treatments. Here we present a new imaging and perfusion device for Arabidopsis roots that provides guidance of the root tip and centering of the root within the chamber to allow symmetric or asymmetric perfusion of the root. By applying treatments specifically to one side of the root we are able to distinguish between cell-autonomous responses and coordinated responses to changing environmental conditions.
INTRODUCTION: A diversity of interactions exists between fungi and other microorganisms, which play central roles in certain human infections and the biological control of plant diseases. However, studying the interplay between filamentous fungi and bacteria using high-resolution microscopies, and monitoring these interactions over a specific time period is technically challenging, since co-inoculation methods that are used at present have two major drawbacks: they either allow the study of interactions at the macroscale over time or on a cellular level at one specific time point. One major problem is due to the polarised growth of filamentous fungi. Hyphae form a three-dimensional interconnected network, which quickly leads to an overgrown system and therefore makes it challenging to follow a single hypha or hyphal compartment. METHODS: Photolithography techniques  were used to produce microfluidic devices made from poly(dimethylsiloxane) (PDMS). Using these microfluidic platforms, individual hyphae of a filamentous fungus can grow into medium-filled microchannels from an agar plug that is placed next to a lateral opening. The height of the microchannels restricts the hyphae to a two-dimensional network. RESULTS: We have developed two microfluidic platforms that overcome the aforementioned problems, namely, the bacterial-fungal interaction (BFI) device and the fluid exchange device. These devices were then used to study the interaction of Coprinopsis cinerea with two Bacillus subtilis strains (168 and NCIB 3610). In the BFI device, motile bacteria can be introduced into the microchannels through a separate inlet and are able to move freely within the channels. Morphological changes of hyphae, physical interactions and the distribution of bacteria in the channels can be observed using high-resolution microscopy at the single cell level over long lengths of time. The fluid exchange device allows rapid exchange of the fluid surrounding the hyphae. Using this platform, the response of hyphae to different compounds can be studied readily. We observed a differential attachment of B. subtilis to C. cinerea hyphae. Further, it was found that B. subtilis NCIB 3610 produces a compound that leads to formation of blebs and emptying of hyphae. DISCUSSION & CONCLUSIONS: Our microfluidic approach offers a novel method to study cellular interactions that are imposed by nature. Specifically, they have been used to probe the interaction of C. cinerea with B. subtilis, providing – for the first time – new insights into this interaction at a cellular level and in real-time. The dynamics of the attachment and movement of the bacteria will be further studied in time and space using fluorescence microscopy. REFERENCES:  D. Hogan; 2002; Science; 296:2229-2232.  D. Duffy; 1998; Anal. Chem.; 70:4974-4984.
Urinary catheters are the major source of hospital infections worldwide and the second most common cause for bloodstream infections. Biofilm formation begins by the initial adherence of bacteria to the catheter surface that build a polysaccharide matrix. The internal communication is based on quorum sensing, involving small signaling molecules such as acyl-homoserine lactone (AHL). As part of the 2007 iGEM competition, we combined the principles of synthetic biology and the engineering cycle to produce ‘Infector Detector’. The design of this DNA construct, based on the MIT Biobrick Registry part F2620, consists of 2 main elements. The first is a constitutive Tet promoter, generating transcription factor LuxR. This binds to AHL, from the biofilm, providing the input of our biosensor system. The resulting AHL-LuxR complex activates the second part, a Lux promoter generating a fluorescent signal in the form of GFPmut3b, giving a detectable output. To avoid bacterial exposure in the clinical scenario, the biobrick was incorporated in an S30 E.Coli cell extract chassis - an in vitro transcription/translation system - as opposed to commonly used E.Coli chassis. Watch the presentation: http://scpro.streamuk.com/uk/player/Default.aspx?wid=8989&ptid=22&t=0