Standardization of Fluorescent Reporter Assays in Synthetic Biology across the Visible Light Spectrum.

In synthetic biology, Fluorescent reporters are frequently used to characterize the expression levels obtained from both genetic parts such as promoters and ribosome binding sites as well as from complex genetic circuits. To this end, plate readers offer an easy and high-throughput way of characterizing both the growth and fluorescence expression levels of cell cultures. However, despite the similar mode of action used in different devices, their output is not comparable due to intrinsic differences in their setup.

Molecular mechanisms of regulation by a β-alanine-responsive Lrp-type transcription factor from Acidianus hospitalis.

The leucine-responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well-represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp-type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non-proteinogenic amino acid β-alanine.

Structural basis of DNA binding by YdaT, a functional equivalent of the CII repressor in the cryptic prophage CP-933P from Escherichia coli O157:H7.

YdaT is a functional equivalent of the CII repressor in certain lambdoid phages and prophages. YdaT from the cryptic prophage CP-933P in the genome of Escherichia coli O157:H7 is functional as a DNA-binding protein and recognizes a 5'-TTGATTNAATCAA-3' inverted repeat. The DNA-binding domain is a helix-turn-helix (HTH)-containing POU domain and is followed by a long α-helix (α6) that forms an antiparallel four-helix bundle, creating a tetramer.

Unlocking the bacterial domain for industrial biotechnology applications using universal parts and tools.

Synthetic biology can play a major role in the development of sustainable industrial biotechnology processes. However, the development of economically viable production processes is currently hampered by the limited availability of host organisms that can be engineered for a specific production process. To date, standard hosts such as Escherichia coli and Saccharomyces cerevisiae are often used as starting points for process development since parts and tools allowing their engineering are readily available.

Separation and Characterization of Protein-DNA Complexes by EMSA and In-Gel Footprinting.

In-gel footprinting enables the precise identification of protein binding sites on the DNA after separation of free and protein-bound DNA molecules by gel electrophoresis in native conditions and subsequent digestion by the nuclease activity of the 1,10-phenanthroline-copper ion [(OP)-Cu] within the gel matrix. Hence, the technique combines the resolving power of protein-DNA complexes in the electrophoretic mobility shift assay (EMSA) with the precision of target site identification by chemical footprinting. This approach is particularly well suited to characterize distinct molecular assemblies in a mixture of protein-DNA complexes and to identify individual binding sites within composite operators, when the concentration-dependent occupation of binding sites, with a different affinity, results in the generation of complexes with a distinct stoichiometry and migration velocity in gel electrophoresis.

In Vivo Screening Method for the Identification and Characterization of Prokaryotic, Metabolite-Responsive Transcription Factors.

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion.

Engineering transcriptional regulation in Escherichia coli using an archaeal TetR-family transcription factor.

Synthetic biology requires well-characterized biological parts that can be combined into functional modules. One type of biological parts are transcriptional regulators and their cognate operator elements, which enable to either generate an input-specific response or are used as actuator modules. A range of regulators has already been characterized and used for orthogonal gene expression engineering, however, previous efforts have mostly focused on bacterial regulators.

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization.

Bacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini-Tn7-lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro- and micro-scale imaging techniques. We demonstrate that a dual bioluminescence-fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro- and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris).

Competitive Repression of the Operon for Arginine and Ornithine Transport by Arginine Repressor and Leucine-Responsive Regulatory Protein in .

Two out of the three major uptake systems for arginine in are encoded by the - gene cluster. ArtJ is the high-affinity periplasmic arginine-specific binding protein (ArgBP-I), whereas encodes the arginine and ornithine periplasmic binding protein (AO). Both ArtJ and ArtI are supposed to combine with the inner membrane-associated ArtQMP transport complex of the ATP-binding cassette-type (ABC).

Regulation of arginine biosynthesis, catabolism and transport in Escherichia coli.

Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term "repression" was proposed by Vogel (The chemical basis of heredity, The John Hopkins Press, Baltimore, 1957) (in opposition to induction) to describe the relative decrease in acetylornithinase production in Escherichia coli cells upon arginine supplementation, whereas the term "regulon" was coined by Maas and Clark (J Mol Biol 8:365-370, 1964) for the ensemble of arginine biosynthetic genes dispersed over the E. coli chromosome but all subjected to regulation by the trans-acting argR gene product.

Transcription Regulators in Archaea: Homologies and Differences with Bacterial Regulators.

The fitness and survival of prokaryotic microorganisms depends on their ability to adequately respond to environmental changes, sudden stress conditions and metabolic shifts. An important mechanism underlying this response is the regulation of gene expression mediated by transcription factors that are responsive to small-molecule ligands or other intracellular signals. Despite constituting a distinct domain of life from bacteria and harboring a eukaryotic-like basal transcription apparatus, it is well established that archaea have similar transcription factors pointing to the existence of shared ancestral proteins and to the occurrence of inter-domain horizontal gene transfer events.

Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology.

Gene expression occurs in two essential steps: transcription and translation. In bacteria, the two processes are tightly coupled in time and space, and highly regulated. Tight regulation of gene expression is crucial.

A sigma factor toolbox for orthogonal gene expression in Escherichia coli.

Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules.

A novel and versatile dual fluorescent reporter tool for the study of gene expression and regulation in multi- and single copy number.

To unravel intricate mechanisms of gene regulation it is imperative to work in physiologically relevant conditions and therefore preferentially in single copy constructs, which are not always easy to manipulate. Such in vivo studies are generally based on enzymatic assays, microarrays, RNA-seq, qRT-PCR, or multicopy reporter gene systems, frequently with β-galactosidase, luciferase or a fluorescent protein as reporter. Each method has its advantages and shortcomings and may require validation.

Differential protein-DNA contacts for activation and repression by ArgP, a LysR-type (LTTR) transcriptional regulator in Escherichia coli.

ArgP is a LysR-type transcriptional regulator (LTTR) that operates with two effector molecules, lysine and arginine, to differentially regulate gene expression. Effector-free ArgP stimulates transcription of all investigated regulon members, except argO, whereas lysine abolishes this effect. Activation of argO, encoding an exporter for arginine and canavanine, is strictly dependent on arginine-bound ArgP.

Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in Escherichia coli.

RutR is a member of the large family of TetR transcriptional regulators in Escherichia coli. It was originally discovered as the regulator of the rutABCDEFG operon encoding a novel pathway for pyrimidine utilization, but its highest affinity target is the control region of the carAB operon, encoding carbamoylphosphate synthase. Unlike most other TetR-like regulators, RutR exerts both positive and negative effects on promoter activity.

The protein-DNA contacts in RutR•carAB operator complexes.

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR.