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Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis
(2013)
The response of Bacillus licheniformis to heat and ethanol stress and the role of the SigB regulon
(2013)
Disruption experiments targeted at the Bacillus licheniformis degSU operon and GFP-reporter analysis provided evidence for promoter activity immediately upstream of degU. pMutin mediated concomitant introduction of the degU32 allele – known to cause hypersecretion in Bacillus subtilis – resulted in a marked increase in protease activity. Application of 5-fluorouracil based counterselection through establishment of a phosphoribosyltransferase deficient Δupp strain eventually facilitated the marker-free introduction of degU32 leading to further protease enhancement achieving levels as for hypersecreting wild strains in which degU was overexpressed. Surprisingly, deletion of rapG – known to interfere with DegU DNA-binding in B. subtilis – did not enhance protease production neither in the wild type nor in the degU32 strain. The combination of degU32 and Δupp counterselection in the type strain is not only equally effective as in hypersecreting wild strains with respect to protease production but furthermore facilitates genetic strain improvement aiming at biological containment and effectiveness of biotechnological processes.
Bacillus subtilis and Bacillus licheniformis are widely used for the large-scale industrial production of proteins. These strains can efficiently secrete proteins into the culture medium using the general secretion (Sec) pathway. A characteristic feature of all secreted proteins is their N-terminal signal peptides, which are recognized by the secretion machinery. Here, we have studied the production of an industrially important secreted protease, namely, subtilisin BPN′ from Bacillus amyloliquefaciens. One hundred seventy-three signal peptides originating from B. subtilis and 220 signal peptides from the B. licheniformis type strain were fused to this secretion target and expressed in B. subtilis, and the resulting library was analyzed by high-throughput screening for extracellular proteolytic activity. We have identified a number of signal peptides originating from both organisms which produced significantly increased yield of the secreted protease. Interestingly, we observed that levels of extracellular protease were improved not only in B. subtilis, which was used as the screening host, but also in two different B. licheniformis strains. To date, it is impossible to predict which signal peptide will result in better secretion and thus an improved yield of a given extracellular target protein. Our data show that screening a library consisting of homologous and heterologous signal peptides fused to a target protein can identify more-effective signal peptides, resulting in improved protein export not only in the original screening host but also in different production strains.
Architecture for platform- and hardware-independent mesh networks : how to unify the channels
(2013)
This paper will prove that mesh networks among different platforms and hardware channels can help to channel valuable information even if public telecommunication infrastructure is not available due to arbitrary reasons. Therefore, results of a simulation for mesh networks on mass events will be provided, followed by the developed architecture and an outlook on future research. The developed architecture is currently being implemented and field tested on mass events.
Our world is well ordered in measurement and number : or why natural constants are as they are
(2013)
All the important natural constants can be logically explained with and derived from the first four ordinal numbers, 1, 2, 3 and 4, its addition to ten and finally the standard values for obviously maximal feasibility Ω and the optimum in our world, the Golden Section (GS), i.e. the number sequences 273 and 618. They both are the first three numbers of irrational results by an arithmetical transformation of simple geometrical relationships by creating multiplicity out of singularity. Both of them show that the infinite is inherent in finiteness and explain in a simple way the smallest deviations and fluctuations between the physical AS-IS state and the obvious spiritual ideal behind: Wherever we look in this world, and especially in important key-positions, we regularly find these sequences. All of the above mentioned numbers so seem to be key players in our world, what can be demonstrated by the derivation of natural constants.
Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0 × 10⁵ cells ml⁻¹. The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.
Shakedown analysis of Reissner-Mindlin plates using the edge-based smoothed finite element method
(2014)
This paper concerns the development of a primal-dual algorithm for limit and shakedown analysis of Reissner-Mindlin plates made of von Mises material. At each optimization iteration, the lower bound of the shakedown load multiplier is calculated simultaneously with the upper bound using the duality theory. An edge-based smoothed finite element method (ES-FEM) combined with the discrete shear gap (DSG) technique is used to improve the accuracy of the solutions and to avoid the transverse shear locking behaviour. The method not only possesses all inherent features of convergence and accuracy from ES-FEM, but also ensures that the total number of variables in the optimization problem is kept to a minimum compared with the standard finite element formulation. Numerical examples are presented to demonstrate the effectiveness of the present method.