Paper-based open microfluidic platform for protein electrophoresis and immunoprobing.

Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

Modeling population dynamics in a microbial consortium under control of a synthetic pheromone-mediated communication system.

Microbial consortia can be used to catalyze complex biotransformations. Tools to control the behavior of these consortia in a technical environment are currently lacking. In the present study, a synthetic biology approach was used to build a model consortium of two Saccharomyces cerevisiae strains where growth and expression of the fluorescent marker protein EGFP by the receiver strain is controlled by the concentration of α-factor pheromone, which is produced by the emitter strain. We have developed a quantitative experimental and theoretical framework to describe population dynamics in the model consortium. We measured biomass growth and metabolite production in controlled bioreactor experiments, and used flow cytometry to monitor changes of the subpopulations and protein expression under different cultivation conditions. This dataset was used to parameterize a segregated mathematical model, which took into account fundamental growth processes, pheromone-induced growth arrest and EGFP production, as well as pheromone desensitization after extended exposure. The model was able to predict the growth dynamics of single-strain cultures and the consortium quantitatively and provides a basis for using this approach in actual biotransformations.

New approaches in bioprocess-control: Consortium guidance by synthetic cell-cell communication based on fungal pheromones.

Bioconversions in industrial processes are currently dominated by single-strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single-strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium-based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell-cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone-responsive gene expression, thereby creating pheromone-dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone-controlled sensor-actor systems, exploiting their unique metabolic properties for microbial consortia approaches.

Pheromone-inducible expression vectors for fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an attractive host for heterologous gene expression. However, expression systems for industrially viable large-scale fermentations are scarce. Several inducible expression vectors for S. pombe have been reported, with the strong thiamine-repressible nmt1+ promoter or derivatives thereof most commonly employed. Previously, the promoter regions of the genes sxa2+ and rep1+ were utilized to couple pheromone signaling to the expression of reporter genes for quantitative assessment of the cellular response to mating pheromones. Here, we exploit these promoters to serve as highly effective, plasmid-based inducible expression systems for S. pombe. Simply by adding synthetic P-factor pheromone, both promoters conferred 50-60% higher peak expression levels than the nmt1+ promoter. Full induction was significantly faster than observed for nmt1+-based expression platforms. Furthermore, the sxa2+ promoter showed very low basal activity and an overall 584-fold induction by synthetic P-factor pheromone. The dose-response curves of both promoters were assessed, providing the opportunity for facile tuning of the expression level by modulating P-factor concentration. Since the expression plasmids relying on the sxa2+ and rep1+ promoters require neither medium exchange nor glucose/thiamine starvation, they proved to be very convenient in handling. Hence, these expression vectors will improve the palette of valuable genetic tools for S. pombe, applicable to both basic research and biotechnology.

Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

Artificial cell-cell communication as an emerging tool in synthetic biology applications.

Cell-cell communication is a widespread phenomenon in nature, ranging from bacterial quorum sensing and fungal pheromone communication to cellular crosstalk in multicellular eukaryotes. These communication modes offer the possibility to control the behavior of an entire community by modifying the performance of individual cells in specific ways. Synthetic biology, i.e., the implementation of artificial functions within biological systems, is a promising approach towards the engineering of sophisticated, autonomous devices based on specifically functionalized cells. With the growing complexity of the functions performed by such systems, both the risk of circuit crosstalk and the metabolic burden resulting from the expression of numerous foreign genes are increasing. Therefore, systems based on a single type of cells are no longer feasible. Synthetic biology approaches with multiple subpopulations of specifically functionalized cells, wired by artificial cell-cell communication systems, provide an attractive and powerful alternative. Here we review recent applications of synthetic cell-cell communication systems with a specific focus on recent advances with fungal hosts.

A yeast pheromone-based inter-species communication system.

We report on a pheromone-based inter-species communication system, allowing for a controlled cell-cell communication between the two species Saccharomyces cerevisiae and Schizosaccharomyces pombe as a proof of principle. It exploits the mating response pathways of the two yeast species employing the pheromones, α- or P-factor, as signaling molecules. The authentic and chimeric pheromone-encoding genes were engineered to code for the P-factor in S. cerevisiae and the α-factor in S. pombe. Upon transformation of the respective constructs, cells were enabled to express the mating pheromone of the opposite species. The supernatant of cultures of S. pombe cells expressing α-factor were able to induce a G1 arrest in the cell cycle, a change in morphology to the typical shmoo effect and expression driven by the pheromone-responsive FIG1 promoter in S. cerevisiae. The supernatant of cultures of S. cerevisiae cells expressing P-factor similarly induced cell cycle arrest in G1, an alteration in morphology typical for mating as well as the activation of the pheromone-responsive promoters of the rep1 and sxa2 genes in a pheromone-hypersensitive reporter strain of S. pombe. Apparently, both heterologous pheromones were correctly processed and secreted in an active form by the cells of the other species. Our data clearly show that the species-specific pheromone systems of yeast species can be exploited for a controlled inter-species communication.