Field-based multiplex and quantitative assay platforms for diagnostics

The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field

Analysis of the behaviour of Dictyostelium discoideum in immobilised state by means of continuous cultivation

Beshay U, Friehs K, Azzam AEM, Flaschel E. Analysis of the behaviour of Dictyostelium discoideum in immobilised state by means of continuous cultivation. BIOPROCESS AND BIOSYSTEMS ENGINEERING. 2003;26(2):117-122.The social amoeba Dictyostelium discoideum is amenable to cultivation in the immobilised state most simply by colonisation of porous supports. An analysis of the growth behaviour of D. discoideum in the immobilised state is reported. For this purpose, D. discoideum was cultivated in continuously operated reactors in a suspension culture (homogeneous system) and immobilised on a porous support (heterogeneous system). Thus, it is possible to compare homogeneous and heterogeneous systems under steady-state conditions. Immobilisation was achieved by the colonisation of porous glass beads (SIRAN). Simple models are applied in order to describe the growth behaviour of fractions of both the cells in free suspension and the cells inside the porous carrier. This analysis shows that D. discoideum inside the pores grows at a rate of only about 10% compared with that in free solution. The consequence of this behaviour is discussed in terms of reactor performance

Cultivation of Dictyostelium discoideum in immobilized form by colonization of porous supports

Beshay U, Friehs K, Azzam AEM, Flaschel E. Cultivation of Dictyostelium discoideum in immobilized form by colonization of porous supports. PROCESS BIOCHEMISTRY. 2003;38(11):1521-1529.Dictyostelium discoideum is a social amoeba. It shows promising characteristics as an expression system for the production of recombinant glycoproteins owing to its ability to perform a whole spectrum of post-translational modifications. However, its application is seriously affected by slow growth rates as well as low maximal cell densities. Since the technique of immobilization often is applied in order to achieve high cell concentrations, D. discoideum was cultivated in the presence of different porous supports with the aim of studying its growth in an immobilized state. On a small scale a shake flask cultivation system with external loop was applied for this purpose. On a larger scale a bioreactor was coupled with an external loop of a column containing a fixed bed of the porous support. During growth on standard axenic (soluble) media, cell densities in the pores of the supports were attained, which were more than one magnitude higher than those normally obtained during suspension culture. Electron micrograph images indicate how D. discoideum colonizes porous support particles. (C) 2003 Elsevier Science Ltd. All rights reserved

Improved beta-glucanase production by a recombinant Escherichia coli strain using a zinc-ion supplemented medium

Beshay U, Miksch G, Friehs K, Flaschel E. Improved beta-glucanase production by a recombinant Escherichia coli strain using a zinc-ion supplemented medium. ENGINEERING IN LIFE SCIENCES. 2007;7(3):253-258.In order to investigate the suitability of different metal chelates for affinity chromatography, an expression vector was constructed. It contained a hybrid beta-glucanase as a model protein fused with a His(6)-tag and a secretion cassette providing the ability to secrete beta-glucanase into the culture medium. Supplementation of zinc to the medium led to a rapidly increased expression and release of the target protein into the cultivation medium. Results in respect to the supplementation of the commonly used Terrific Broth "TB-medium" with different metal ions are reported with special emphasis on the influence of zinc ions. A concentration of zinc ions in the order of about 0.175 mM led to optimal results. Batch cultivation under well-controlled conditions showed that the growth behavior did not change significantly by adding zinc ions. Growth in a stirred tank bioreactor was much faster in unsupplemented TB-medium compared to shake flask experiments leading to a much higher biomass concentration (15 g/L instead of 3 g/L). The secretion of beta-glucanase under theses conditions started at the transition into the stationary phase and increased to yield an extracellular activity of 1350 U/mL at the end of the fermentation process. An even higher yield of extracellular beta-glucanase (2800 U/mL) was reached when the fermentation was carried out with TB-medium supplemented with 0.175 mM ZnSO4

Mass production of Dictyostelium discoideum in homogenous and hetergeneous cultivation systems

Lu YH, Beshay U, Friehs K, Flaschel E. Mass production of Dictyostelium discoideum in homogenous and hetergeneous cultivation systems. PROCESS BIOCHEMISTRY. 2004;39(12):1859-1870.The social amoeba Dictyostelium discoideum is a promising host for the expression of recombinant proteins requiring post-translational modifications. Limited maximal cell densities and slow growth rates, however, disfavor its application. Little attention has been paid to improve its cultivation. Here, some strategies are described, which allow the attainment of higher cell densities. This can be achieved by cultivation on an improved synthetic medium as well as in immobilized form. Two promising inorganic porous supports are presented-broken pumice and a ceramic catalyst carrier. Cell densities up to 4.5 x 10(7) ml(-1) are obtained during suspension cultivation on the improved synthetic medium. This is about three times as much as can be expected for cultivations on conventional complex media. Cells in the pores of broken pumice and a ceramic support reach up to 15-20 times higher local cell densities compared with cells growing on conventional axenic media in suspension. Thus, the cell density of 3.5 x 10(8) ml(-1) in the ceramic carrier (CeramTec(R)) is the highest cell density for D. discoideum observed so far in cultivation systems. The immobilized cell density could be maintained for a long period of time by either repeated medium replacement or continuous cultivation. To minimize the external volume of the medium a trickle bed reactor was operated continuously. (C) 2003 Elsevier Ltd. All rights reserved

Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli

Spexard M, Beshay U, Risse JM, Miksch G, Flaschel E. Screening for conditions of enhanced production of a recombinant beta-glucanase secreted into the medium by Escherichia coli. BIOTECHNOLOGY LETTERS. 2010;32(2):243-248.The extracellular production of a hybrid bacterial beta-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a beta-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (> 200 U ml(-1)) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol