A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms

NADPH-generating systems in bacteria and archaea

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms.

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overex- pression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting dif- ferent codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Transmembrane helix prediction and codon usage landscapes for the different variants for DGGGPs.

<p>(a) Transmembrane helix prediction plot depicting the probability of residues being in a transmembrane helix domain (red bars), on the inside or cytosolic side of the membrane (blue line) or outside of the membrane (purple line) ((TMHMM v2.0). Codon usage landscapes are depicted based on Relative Codon Adaptiveness (RCA) scores for individual amino-acids and a moving average over 5 codons (black line), for (b) the wild-type gene for native host codon usage (<i>M</i>. <i>maripaludis</i> C5); (c) the codon-harmonized gene variant for <i>E</i>. <i>coli</i> codon usage; (d) the codon-optimized gene variant for <i>E</i>. <i>coli</i> codon usage (e) the wild-type gene variant for <i>E</i>. <i>coli</i> codon usage.</p

Membrane-integrated production levels for all codon usage variants.

<p>Production levels in <i>E</i>. <i>coli</i> LEMO(DE3) were determined by whole-cell GFP-fluorescence at different transcriptional tuning by varying the L-rhamnose concentration (indicated in μM). All expression experiments were at least performed in biological triplicates.</p

Overview of all 6 tested membrane proteins for which the expression of different gene variants was studied and their analyzed codon usage parameters.

<p>Overview of all 6 tested membrane proteins for which the expression of different gene variants was studied and their analyzed codon usage parameters.</p

Schematic representation of the flow device.

<p>A) Schematic representation of the flow device, with the dimensions in mm. Depicted in red and blue are the in- and outflow channels of the top compartment (light green). The respective in- and outflow channels of the lower compartment (yellow) are given in purple and dark green. B) Electron microscopy image of a microsieve. C) Electron microscopy image of a microdish. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036982#pone.0036982.s001" target="_blank">File S1</a> for more views of the device.</p

Fluorescent nematodes observed in the flow device.

<p>A) Nematodes floating over the wells while the chamber is filled with liquid. The fluorescent oesophagus in the front side of the nematode is clearly visible. B) Nematode trapped in a well filled with fluorescent <i>E. coli</i> cells after removing the liquid from the chamber. C) Next day: A nematode after consuming all fluorescent bacteria from the well, resulting in observable fluorescence in the nematode intestine.</p