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Robust synthetic biology applications rely heavily on the design and assembly of DNA parts with specific functionalities based on engineering principles. However, the assembly standards adopted by different communities vary considerably, thus limiting the interoperability of parts, vectors and methods. We hereby introduce the SEVA 3.1 platform consisting of the SEVA 3.1 vectors and the Golden Gate‐based ‘SevaBrick Assembly’. This platform enables the convergence of standard processes between the SEVA platform, the BioBricks and the Type IIs‐mediated DNA assemblies to reduce complexity and optimize compatibility between parts and methods. It features a wide library of cloning vectors along with a core set of standard SevaBrick primers that allow multipart assembly and exchange of short functional genetic elements (promoters, RBSs) with minimal cloning and design effort. As proof of concept, we constructed, among others, multiple sfGFP expression vectors under the control of eight RBSs, eight promoters and four origins of replication as well as an inducible four‐gene operon expressing the biosynthetic genes for the black pigment proviolacein. To demonstrate the interoperability of the SEVA 3.1 vectors, all constructs were characterized in both Pseudomonas putida and Escherichia coli. In summary, the SEVA 3.1 platform optimizes compatibility and modularity of inserts and backbones with a cost‐ and time‐friendly DNA assembly method, substantially expanding the toolbox for successful synthetic biology applications in Gram‐negative bacteria.

As synthetic biology moves away from trial and error and embraces more formal processes, workflows have emerged that cover the roadmap from conceptualization of a genetic device to its construction and measurement. This latter aspect (i.e. characterization and measurement of synthetic genetic constructs) has received relatively little attention thus far, but it is crucial for their outcome. An end-to-end use case for engineering a simple synthetic device is presented which is supported by information standards and computational methods, and which focuses on such characterization/measurement. This workflow captures the main stages of genetic device design and description and offers standardized tools for both population-based measurement and single-cell analysis. To this end, three separate aspects are addressed. First, the specific vector features. Although device/circuit design has been successfully automated, important structural information is usually overlooked, as is the case of plasmid vectors. The use of the Standard European Vector Architecture (SEVA) is advocated for selecting the optimal carrier of a design and its thorough description, in order to unequivocally correlate digital definitions and molecular devices. A digital version of this plasmid format was developed with the Synthetic Biology Open Language (SBOL) along with a software tool that allows users to embed genetic parts in vector cargoes. This enables annotation of a mathematical model of the device's kinetic reactions formatted with the Systems Biology Markup Language (SBML). From that point onwards the experimental results and their in silico counterparts proceed alongside, with constant feedback to preserve consistency between them. A second aspect involves a framework for the calibration of fluorescence-based measurements. One of the most challenging endeavors in standardization, metrology, is tackled by reinterpreting the experimental output in light of simulation results, allowing us to t

BACKGROUND: Signal transduction plays a fundamental role in the understanding of cellular physiology. The bacterialphosphotransferase system (PTS) together with the PEP/pyruvate node in central metabolism represents asignaling unit that acts as a sensory element and measures the activity of the central metabolism.Pseudomonas putida possesses two PTS branches, the C-branch (PTSFru) and a second branch (PTSNtr),which communicate with each other by phosphate exchange. Recent experimental results showed a cross talkbetween the two branches. However, the functional role of the crosstalk remains open. RESULTS: A mathematical model was set up to describe the available data of the state of phosphorylation of PtsN, one ofthe PTS proteins, for different environmental conditions and different strain variants. Additionally, data fromflux balance analysis was used to determine some of the kinetic parameters of the involved reactions. Based onthe calculated and estimated parameters, the flux distribution during growth of the wild type strain on fructosecould be determined. CONCLUSION: Our calculations show that during growth of the wild type strain on the PTS substrate fructose, the major partof the phosphoryl groups is provided by the second branch of the PTS. This theoretical finding indicates a newrole of the second branch of the PTS and will serve as a basis for further experimental studies

Metal working fluids (MWF) have been employed in industry since the time of the Egyptians, for cooling and lubrication during cutting and grinding of metal. There are several types, which can be broadly categorized as oil-based, semisynthetic, and synthetic. It is estimated that in the region of 5 × 109 L−1 are in use in the European Union (EU) at any one time,where they represent in the region of 16% of manufacturing industries costs. The microbiology of MWF is of interest for two contrasting reasons namely: (1) they represent attractive nutrient sources for microorganisms, which lead to bio-deterioration, reducing metal working performance and potentially leading to invasion of human pathogens; (2) in contrast, the same biodegradation activities of potential microbial colonizers can be harnessed and exploited for biotreatment of waste fluids. These contrasting microbiological issues are discussed and details provided. The need for research to develop new MWF formulations that are unattractive to microbial colonization when in use, but with a few chemical modifications can be rendered susceptible to microbial biodegradation, when subsequent disposal of waste fluids is required, is discussed.

Microorganisms play a fundamental role in the cycle of nutrients and energy on our planet. The characterization of microorganisms in their natural environment is the objective and the greatest challenge of microbial ecologists. Single-cell techniques havewww.lw20.com become an integral part in the environmental microbiologist’s toolbox. One of these methods, Fluorescence in situ Hybridization (FISH), is a phylogenetic staining using labeled nucleotide probes to target ribosomal RNA of microbial cells in fixed samples.

This chapter attempts to show how the mycelia of filamentous fungi can act as transport vectors for contaminants and contaminant-degrading bacteria and hence influence the biodegradation of (hydrophobic) organic chemicals in the subsurface. It establishes a conceptual framework of the influence of fungus-mediated transport processes on the bioaccessibility of subsurface-contaminants and discusses them as interplay between microbial ecology and physical constraints.

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Microbial communities in their fully hydrated state can be imaged in space and time (4-dimensionally) by laser scanning microscopy using 1-photon or 2-photon excitation. In this chapter we provide guidance on how to examine microorganisms associated with the degradation of liquid, viscous and solid hydrocarbons. The details of mounting samples for upright and inverted microscopes as well as options for fluorescence staining of bacteria and hydrocarbons are presented. Then suggestions are made for recording images and subsequent digital image analysis. Finally practical aspects are discussed such as the material and software needed, the time required and a guide for trouble shooting.

Bacteria occur as dynamic and diverse populations and communities in nature. To understand bacterial ecology in natural environments, new techniques are required for answering questions which could not be addressed by traditional methods alone. Now, more than ever, flow cytometric analysis and cell sorting of sub-populations and sub-communities,?especially of ones not yet culturable, have been recognized as valuable tools for microbiologists that provide unique opportunities to investigate and quantify various aspects of micro-environmental processes by combining them with the Omics technologies.

The protocol describes the analysis and data interpretation of membrane phospholipid fatty acid (PLFA) profiles as biomarkers for the characterization of microbial communities in environmental samples with special emphasis to soils and sediments contaminated with organic hydrocarbons. This simple method in microbial ecology studies provides an estimate of the viable microbial biomass, gives first indications for the structure of the microbial community, as well as can inform about the physiological status of the microbiota. In environmental biotechnological applications, PLFA analysis can be used for monitoring of bioremediation techniques by presenting indicators for existing biological activities and for microbiota changes caused by the treatment.

A rather efficient solvent adaptation mechanism enabling several gram-negative bacteria to tolerate and grow in the presence of membrane-disturbing compounds is the isomerization of cis- to trans-unsaturated membrane fatty acids. The degree of isomerization obviously depends on the toxicity and the concentration of membrane-affecting agents. Synthesis of trans-fatty acids comes about by direct isomerization of the respective cis-configuration of the double bond without shifting the position. The purpose of the conversion of the cis-configuration to trans is apparently the rapid adaptation of the membrane fluidity to rising temperature or the presence of toxic organic hydrocarbons. The cis-trans-isomerase (Cti) is a constitutively expressed periplasmic enzyme that - to exert its action - necessitates neither ATP nor other cofactors, and consistently, is independent of de novo synthesis of lipids. A heme-binding site typical of cytochrome c-type proteins is present in the predicted Cti polypeptide indicating a reaction mechanism that renounces temporary saturation of the double bond. Due to its direct correlation with toxicity, cis-trans-isomerization is a potential biomarker for recording solvent stress or changes of other environmental conditions.

This chapter attempts to show how the hydrophobicity of hydrocarbons influences their availability for toxic exposure, microbial degradation and growth, and how it can control the carrying capacity of a microbial habitat. Bioavailability is operationalized and presented as a process at the interface between biological dynamics and physicochemical constraints.

In recent years, the use of compound specific isotope analysis (CSIA) to measure stable isotope fractionation developed into one of the most important tools for assessing biodegradation of aromatic hydrocarbons in contaminated groundwater. Biodegradation reactions are often accompanied by shifts in the stable isotope ratios of the naturally abundances of e.g., 13C/12C, 2H/1H, or other elements of the compound of interest. This isotope fractionation is measured in the residual substrate fraction sampled from monitoring wells by means of CSIA. If laboratory degradation experiments have been performed to obtain stable isotope fractionation factors for a given compound and reaction one can use the Rayleigh equation to calculate the extent of biodegradation in the environment. The big advantage of the stable isotope fractionation approach is that one can even quantify the extent of biodegradation in extremely complex matrices such as aquifers. Several field studies successfully showed astonishing accuracy of the method to determine biodegradation rates on contaminated field sites. Nowadays, stable isotope tools are widely accepted by authorities as a reliable tool to prove biodegradation in natural attenuation. Here, we give a brief introduction how to use CSIA for assessing stable isotope fractionation of aromatic hydrocarbons together with a few selected successful examples of application.

A concept of in situ microcosms, which allows analyzing structure, function, and activity of microbial communities in situ, as well as the enrichment of microorganisms for subsequent cultivation in laboratory experiments is described. The concept is based on the application of stable isotope-labeled compounds supplied within the microcosm system. Microorganisms capable of utilizing these substrates as carbon or nitrogen sources become labeled upon metabolism and biosynthesis. Stable isotope probing (SIP) of biomarker molecules such as?fatty acids, proteins, or nucleic acids allows tracing the flux of the label into the biomass and?offers potential for linking structure and function of the active microbial communities.

Fate and transport of hydrophobic organic compounds (HOC) are strongly influenced by their interactions with environmental matrices. These interactions can be grouped into those of non-aqueous phase liquids (NAPL), i.e., spreading of oil on solid surfaces and its movement in porous media and those of predominantly water-dissolved molecules which may sorb onto solids or partition into organic matter or NAPL phases. All these types of sequestration phenomena lead to reduced contact of water-borne organisms with the HOC. In contrast, sorption to mobile matrices may mobilize HOC and thus increase bioavailability and toxicological risk.

Three approaches for the determination of the hydrophobicity of microorganisms and/or their tendency to physically interact with hydrocarbon phases are presented. These include the bacterial/microbial adhesion to hydrocarbon (BATH/MATH)-test, hydrophobic interaction chromatography and the contact angle measurements of microbial cell mats.

The protocol describes the application of the membrane adaptive mechanism of Gram-negative bacteria belonging to the genera Pseudomonas and Vibrio, the isomerization of?cis- to trans-unsaturated membrane fatty acids as a tool for the measurement of the toxicity of?membrane disturbing compounds. The degree of isomerization directly depends on toxicity and concentration of membrane affecting agents. Synthesis of trans fatty acids is apparent within 30 min after addition of stressors by direct isomerization of the respective cis configuration of the double bond without shifting the position. The purpose of the conversion of the cis configuration to trans is apparently a rapid decrease of the membrane fluidity to rising temperature or the presence of toxic organic hydrocarbons. Therefore, for those bacteria in?which this mechanism is present it offers the possibilities to use the trans/cis ratio of unsaturated fatty acids as an elegant, reliable, and rapid bioindicator for membrane stress in experimental setups.

Several classes of organic compounds are toxic for living organisms as they accumulate in and disrupt cell membranes. In these cases, the dose-dependent toxicity of a compound correlates with the logarithm of its partition coefficient between octanol and water (logP). Substances with a logP value between 1 and 5 are, in general, toxic for whole cells. Therefore, toxic effects of hydrocarbons on microorganisms can cause problems in bioremediation of highly contaminated sites. The toxic effect of most hydrocarbons is caused by general, nonspecific effects on membrane fluidity due to their accumulation in the lipid bilayer. Only exceptions are hydrocarbons with specific chemically active functional groups such as aldehydes and epoxides that show an additional chemical toxicity. Most compounds with a higher hydrophobicity than logP of 4 such as e.g., alkanes, PAHs, and biphenyl(s) have very low water solubility, thus their bioavailability is too low to show a toxic effect. By combining the logP value with the water solubility of a compound the maximum membrane concentration (MMC) of a compound can be calculated. By using this parameter it is possible to predict the potential toxicity even of unknown hydrocarbons.

Many toxic hydrocarbons that are present as environmental pollutants are potential substrates for bacteria; other, very hydrophobic hydrocarbons exhibit extremely low water solubility and are poorly bioavailable. The development of specific adaptive mechanisms to the toxicity as well as the low bioavailability of these substrates allows many bacteria to cope with such challenges. Strategies of bacteria to increase the accessibility of these compounds are modifications of their cell surfaces or the release of biosurfactants. Both "strategies" aim at an increased accessibility of the compounds, either by the reduction of surface tension or by allowing a direct hydrophobic-hydrophobic interaction between cell surface and the substrates. The toxicity of hydrocarbons is mainly caused by their permeabilizing effect on the cytoplasmic membranes leading also to a loss of ATP and a decrease in the proton gradient. Bacteria are able to modify their cellular energetics in order to adapt to the presence of toxic hydrocarbons by activating their electron transport phosphorylation systems allowing homeostasis of ATP level and energy charge in the presence of the toxic conditions, however, at the price of a reduced growth yield.

Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction, of the cellular arsenic was present as the glutathione conjugate As(GS)

sigma(54) is unique among the bacterial sigma factors. Besides not being related in sequence with the rest of such factors, its mechanism of transcription initiation is completely different and requires the participation of a transcription activator. In addition, whereas the rest of the alternative sigma factors use to be involved in transcription of somehow related biological functions, this is not the case for sigma(54) and many different and unrelated genes have been shown to be transcribed from sigma(54)-dependent promoters, ranging from flagellation, to utilization of several different carbon and nitrogen sources, or alginate biosynthesis. These genes have been characterized in many different bacterial species and, only until recently with the arrival of complete genome sequences, we have been able to look at the sigma(54) functional role from a genomic perspective. Aided by computational methods, the sigma(54) regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae. Here we present the analysis of the sigma(54) regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440. We have developed an improved method for the prediction of sigma(54)-dependent promoters which combines the scores of sigma(54)-RNAP target sequences and those of activator binding sites. In combination with other evidence obtained from the chromosomal context and the similarity with closely related bacteria, we have been able to predict more than 80% of the sigma(54)-dependent promoters of P. putida with high confidence. Our analysis has revealed new functions for sigma(54) and, by means of comparative analysis with the previous studies, we have drawn a potential mechanism for the evolution of this regulatory system.