A novel system for spatial and temporal imaging of intrinsic plant water use efficiency

Instrumentation and methods for rapid screening and selection of plants with improved water use efficiency are essential to address current issues of global food and fuel security. A new imaging system that combines chlorophyll fluorescence and thermal imaging has been developed to generate images of assimilation rate (A), stomatal conductance (gs), and intrinsic water use efficiency (WUEi) from whole plants or leaves under controlled environmental conditions. This is the first demonstration of the production of images of WUEi and the first to determine images of gs from themography at the whole-plant scale. Data are presented illustrating the use of this system for rapidly and non-destructively screening plants for alterations in WUEi by comparing Arabidopsis thaliana mutants (OST1-1) that have altered WUEi driven by open stomata, with wild-type plants. This novel instrument not only provides the potential to monitor multiple plants simultaneously, but enables intra- and interspecies variation to be taken into account both spatially and temporally. The ability to measure A, gs, and WUEi progressively was developed to facilitate and encourage the development of new dynamic protocols. Images illustrating the instrument's dynamic capabilities are demonstrated by analysing plant responses to changing photosynthetic photon flux density (PPFD). Applications of this system will augment the research community's need for novel screening methods to identify rapidly novel lines, cultivars, or species with improved A and WUEi in order to meet the current demands on modern agriculture and food production. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology

Exploiting Nucleotide Composition to Engineer Promoters

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines