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One major goal of biology is to provide a quantitative description of cellular physiology. This task is complicated by population effects, which perturb culture conditions and mask the behavior of the individual cell. To overcome these limitations, the construction and operation of a microfluidic bioreactor is presented. The new reactor concept guarantees constant environmental conditions and single cell resolution, thus it was named Envirostat (environment, constant). In the Envirostat, cells are contactless trapped by negative dielectrophoresis (nDEP) and cultivated in a constant medium flow. To control chip temperature, a Peltier device was constructed. Joule heating by nDEP was quantified with Rhodamine B in dependence of applied voltage, field mode, medium conductivity, and flow velocity. The integration of the Joule heating effect in the temperature control allowed setting and maintaining the cultivation temperature. For single cell cultivation of Saccharomyces cerevisiae, medium composition changes below 0.001% were estimated by computational fluid dynamic simulation. These changes were considered not to influence cell physiology. Finally, single S. cerevisiae cells were cultivated for more than four generations in the Envirostat, thus showing the applicability of the new reactor concept. The Envirostat facilitates single cell research and might simplify the investigation of hitherto difficult to access biological phenomena such as the true regulatory and physiological response to genetic and environmental perturbations.

Single cell analysis is mainly limited to single time-point measurements, without the possibility to track behavioral changes of a single cell or its descendents. Here, the integration of a spatiotemporal single cell lab-on-a-chip system with an automated cultivation device allows single cell analysis under defined growth conditions and, especially, semiautomated cell retrieval and growth kinetic analysis of the single cell descendants. Performance of the new platform was evaluated using the yeast Saccharomyces cerevisiae. The yeast was singularized in the lab-on-a-chip (Envirostat), which combines the possibility of cell cultivation with cell analysis. Singularized cells were collected in a microtiter plate and cultivated in a semiautomated cultivation device (Bioscreen C). S. cerevisiae showed highly reproducible and glucose concentration independent growth kinetics in population experiments. Yet, growth kinetics in cultures inoculated with only one or few cells exhibited strong variations, because of an unexpected growth phenotype: colony formation in submerse cultures. Interestingly, the colony-like structures grew for more then 60 h and were stable for at least 82 h, despite rigorous shaking. Cell agglomeration due to pseudohyphal growth could be excluded, suggesting changes in cell wall properties of yeast populations starting from a single yeast. Single cell analysis still exhibits unexpected obstacles. Nevertheless, this new single cell analysis platform can be used for studying cellular dynamics of single cells and expanded cell populations thereof. '

We present a prototype for a universal world-to-chip interface for electrical and fluidic connections to lab-on-a-chip devices. The concept is based on spring supported connections to secure and define the contact force for each wire and tubing. We demonstrate the functionality of a manual system and propose the design of an automated system. The new interface provides several useful characteristics: A reliable and reversible leak-free connection is rapidly achieved. The fluidic interface sealed a PMMA chip up to a maximal pressure of 2,070 kPa and reliably connected a six port glass chip. Damage during chip assembly is prevented by an easy handling procedure in combination with adjustable and reproducible contact forces. Destructive fluid overpressure during chip operation is avoided by the relief valve functionality of the interface. The compression based setup is easily adaptable to other chip designs. It is biocompatible and can be used for analytical measurements due to the exclusive use of certified materials and its adhesive free design. The new interface overcomes one of the main obstacles in miniaturization, the connection of the lab-on-a-chip device with the macro world.

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The research field of single cell analysis is rapidly expanding, driven by developments in flow cytometry, microscopy, lab-on-a-chip devices, and many other fields. The promises of these developments include deciphering cellular mechanisms and the quantification of cell-to-cell differences, ideally with spatio-temporal resolution. However, these promises are challenging as the analytical techniques have to cope with minute analyte amounts and concentrations. We formulate first these challenges and then present state-of-the-art analytical techniques available to investigate the different cellular hierarchies--from the genome to the phenome, i.e., the sum of all phenotypes.

We present the design, manufacturing, and characterization of a novel miniaturized on-tube-seal configuration for microfluidic devices. The seal is based on a previously developed world-to-chip spring-based interface by Kortmann et al. (Lab Chip 9:1455-1460, 2009a, which enables rapid and reliable microfluidic connections. In this study, the dead volumes, the contact pressure, the discontinuous fluidic-profile transmission, and the space requirements were significantly optimized by a new on-tube-seal configuration. Maintaining the advantages of the previously described interface, the new on-tube-seal configuration has a dead volume of only 18 nl, withstands pressures higher than 2,800 kPa with only 3.8 N applied contact force, and allows continuous capillary to chip fluidic profile transmission. The on-tube-seal configuration consists of a miniaturized o-ring (0.5 9 0.3 mm) integrated into a 1/16 00 tubing that reduces space requirements to a minimal sealing grid of 1.59 mm and is easily adaptable to any planar channel opening of micro fluidic devices. In summary, we present a novel combination of gasket and tubing, which we termed on-tube-seal that allows simple, rapid, and reliable world-to-chip sealing.

der L-Lysinausbeute von C. glutamicum aus. Die beiden Promotoren des Citratsynthasegens gltA wurden mitsamt ihrer regulatorischen Regionen gegen Promotoren deutlich reduzierter Stärke aus einer dapA-Promotorkollektion ersetzt. Als Ergebnis standen neun Stämme mit graduell reduzierter CS-Aktivität zur Verfügung mit einer Bandbreite von 32± 6 % der CS-Aktivität des Ausgangsstammes. Die Charakterisierung dieser Stämme zeigte, dass bereits die Reduktion auf 32 % der CS-Aktivität in einer signifikanten Steigerung der Lysinausbeute resultiert. Die Stämme mit deutlich stärker reduzierter CS-Aktivität zeigten noch höhere Lysinausbeuten. Bei 6 % der ursprünglichen CS-Aktivität wurde eine Lysinausbeute von 0,32 g/g festgestellt, was gegenüber dem Referenzstamm einer 88 %igen Steigerung entspricht. Gleichzeitig wurde eine deutliche Abnahme der Wachstumsrate festgestellt. Diese Korrelation zeigt, dass die CS als ein Schalter zwischen Bildung von Biomasse auf der einen und Lysinbildung auf der anderen Seite genutzt werden kann. Die Reduktion der CS-Aktivität auf 10 % in einem besseren Lysinproduzenten führte zu einem Stamm mit einer Lysinausbeute von 0,5 g/g, was die höchste aller bislang publizierten Lysinausbeuten darstellt.