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Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.
Against the background of growing data in everyday life, data processing tools become more powerful to deal with the increasing complexity in building design. The architectural planning process is offered a variety of new instruments to design, plan and communicate planning decisions. Ideally the access to information serves to secure and document the quality of the building and in the worst case, the increased data absorbs time by collection and processing without any benefit for the building and its user. Process models can illustrate the impact of information on the design- and planning process so that architect and planner can steer the process. This paper provides historic and contemporary models to visualize the architectural planning process and introduces means to describe today’s situation consisting of stakeholders, events and instruments. It explains conceptions during Renaissance in contrast to models used in the second half of the 20th century. Contemporary models are discussed regarding their value against the background of increasing computation in the building process.
Suspension depletion approach for exemption of infected Solanum jasminoides cells from pospiviroids
(2018)
Despite numerous studies, viroid elimination from infected plants remains a very challenging task. This study introduces for the first time a novel ‘suspension depletion’ approach for exemption of Solanum jasminoides plants from viroids. The proposed method implies initial establishment of suspension cultures of the infected plant cells. The suspended cells were then physically treated (mild thermotherapy, 33 °C), which presumably delayed the replication of the viroid. The viroid concentration in the treated biomass was monitored weekly using pospiviroid-specific PCR. After 10–12 weeks of continuous treatment, a sufficient decrease in viroid concentration was observed such that the infection became undetectable by PCR. The treated single cells then gave rise to microcolonies on a solid culture medium and the obtained viroid-negative clones were further promoted to regenerate into viroid-free plants. Three years of accumulated experimental data suggests feasibility, broad applicability, and good efficacy of the proposed approach.