Synthetic biology and microdevices : a powerful combination

Recent developments demonstrate that the combination of microbiology with micro-and nanoelectronics is a successful approach to develop new miniaturized sensing devices and other technologies. In the last decade, there has been a shift from the optimization of the abiotic components, for example, the chip, to the improvement of the processing capabilities of cells through genetic engineering. The synthetic biology approach will not only give rise to systems with new functionalities, but will also improve the robustness and speed of their response towards applied signals. To this end, the development of new genetic circuits has to be guided by computational design methods that enable to tune and optimize the circuit response. As the successful design of genetic circuits is highly dependent on the quality and reliability of its composing elements, intense characterization of standard biological parts will be crucial for an efficient rational design process in the development of new genetic circuits. Microengineered devices can thereby offer a new analytical approach for the study of complex biological parts and systems. By summarizing the recent techniques in creating new synthetic circuits and in integrating biology with microdevices, this review aims at emphasizing the power of combining synthetic biology with microfluidics and microelectronics

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND:Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS:The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION:This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism

Methodology and ontology in microbiome research

Research on the human microbiome has gen- erated a staggering amount of sequence data, revealing variation in microbial diversity at the community, species (or phylotype), and genomic levels. In order to make this complexity more manageable and easier to interpret, new units—the metagenome, core microbiome, and entero- type—have been introduced in the scientific literature. Here, I argue that analytical tools and exploratory statisti- cal methods, coupled with a translational imperative, are the primary drivers of this new ontology. By reducing the dimensionality of variation in the human microbiome, these new units render it more tractable and easier to interpret, and hence serve an important heuristic role. Nonetheless, there are several reasons to be cautious about these new categories prematurely ‘‘hardening’’ into natural units: a lack of constraints on what can be sequenced metagenomically, freedom of choice in taxonomic level in defining a ‘‘core microbiome,’’ typological framing of some of the concepts, and possible reification of statistical constructs. Finally, lessons from the Human Genome Project have led to a translational imperative: a drive to derive results from the exploration of microbiome variation that can help to articulate the emerging paradigm of per- sonalized genomic medicine (PGM). There is a tension between the typologizing inherent in much of this research and the personal in PGM

Inferences from inventories of microbes in ecological vineyard settings

The effect of degraded soil conditions on microbial communities associating the rhizosphere and roots of grapevines was investigated within the frame of project CORE Organic Plus ReSolVe. Several relationships based on Dice cluster analyses of DGGE gel profiles suggest that fungal and bacterial communities from degraded and non-degraded areas differ. Results also suggest that composted organic amendments had an effect on community structures. However, the comparison of diversity indexes did not show differences between degraded and non-degraded plots. They suggested rich and even diversity of bacteria and fungi in any of the tested samples. Culture dependent analysis emphasized that a high diversity of Streptomyces spp. associates grapevine roots in degraded and non-degraded areas. Streptomyces species, best known for their potential to produce antibiotics, are increasingly depicted as beneficial plant associated bacteria

Ciliary contact interactions dominate surface scattering of swimming eukaryotes

Interactions between swimming cells and surfaces are essential to many microbiological processes, from bacterial biofilm formation to human fertilization. However, in spite of their fundamental importance, relatively little is known about the physical mechanisms that govern the scattering of flagellated or ciliated cells from solid surfaces. A more detailed understanding of these interactions promises not only new biological insights into structure and dynamics of flagella and cilia, but may also lead to new microfluidic techniques for controlling cell motility and microbial locomotion, with potential applications ranging from diagnostic tools to therapeutic protein synthesis and photosynthetic biofuel production. Due to fundamental differences in physiology and swimming strategies, it is an open question whether microfluidic transport and rectification schemes that have recently been demonstrated for pusher-type microswimmers such as bacteria and sperm cells, can be transferred to puller-type algae and other motile eukaryotes, as it is not known whether long-range hydrodynamic or short-range mechanical forces dominate the surface interactions of these microorganisms. Here, using high-speed microscopic imaging, we present direct experimental evidence that the surface scattering of both mammalian sperm cells and unicellular green algae is primarily governed by direct ciliary contact interactions. Building on this insight, we predict and verify experimentally the existence of optimal microfluidic ratchets that maximize rectification of initially uniform Chlamydomonas reinhardtii suspensions. Since mechano-elastic properties of cilia are conserved across eukaryotic species, we expect that our results apply to a wide range of swimming microorganisms.Comment: Preprint as accepted for publication in PNAS, for published journal version (open access) and Supporting Information see http://dx.doi.org/10.1073/pnas.121054811

Introducing the concept of biocatalysis in the classroom: The conversion of cholesterol to provitamin D 3

Biocatalysis is a fundamental concept in biotechnology. The topic integrates knowledge of several disciplines; therefore, it was included in the course “design and optimization of biological systems” which is offered in the biochemistry curricula. We selected the ciliate tetrahymena as an example of a eukaryotic system with potential for the biotransformation of sterol metabolites of industrial interest; in particular, we focused on the conversion of cholesterol to provitamin D3. The students work with wild type and recombinant strains and learn how sterol pathways could be modified to obtain diverse sterol moieties. During the course the students identify and measure the concentration of sterols. They also search for related genes by bioinformatic analysis. Additionally, the students compare biotransformation rates, growing the ciliate in plate and in a bioreactor. Finally, they use fluorescence microscopy to localize an enzyme involved in biotransformation. The last day each team makes an oral presentation, explaining the results obtained and responds to a series of key questions posed by the teachers, which determine the final mark. In our experience, this course enables undergraduate students to become acquainted with the principles of biocatalysis as well as with standard and modern techniques, through a simple and robust laboratory exercise, using a biological system for the conversion of valuable pharmaceutical moieties.Fil: De Luca, Belén M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nudel, Berta Clara. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gonzalez, Rodrigo Horacio. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Nusblat, Alejandro David. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome

Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin bio-synthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula x Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement