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Engineering bacterial properties requires precision and fine-tuning for optimal control of the desired application. In consequence, it is essential to accurately turn the function of interest from OFF to ON state and vice versa, avoiding any type of residual activation. For this type of purpose, light switches have revealed a clean and powerful tool in which control does not depend on the addition of chemical compounds that may remain in the media. To reach this degree of directed regulation through light, the switch based on the cyanobacterial two-component system CcaSR system was previously adapted to manipulate Pseudomonas putida for transcription of a gene of interest. In this chapter, we describe how to induce biofilm formation by placing the expression of the c-di-GMP-producing diguanylate cyclase PleD from Caulobacter sp. under the control of the CcaSR system. The regulation through optogenetics accomplished with this protocol promotes higher exploitation of biofilm beneficial features in a cheaper and cleaner way compared to chemical induction.

Molecular Biology has long tried to discover mechanisms, considering that unless we understand the principles, we cannot develop applications. Now machine learning and artificial intelligence enable direct leaps to application without understanding the principles. Will this herald a decline in mechanistic studies?

Colony formation is key to many ecological and biotechnological processes. In its early stages, colony formation involves the concourse of a number of physical and biological parameters for generation of a distinct 3D structure-the specific influence of which remains unclear. We focused on a thus far neglected aspect of the process, specifically the consequences of the differential pressure experienced by cells in the middle of a colony versus that endured by bacteria located in the growing periphery. This feature was characterized experimentally in the soil bacterium Pseudomonas putida. Using an agent-based model we recreated the growth of microcolonies in a scenario in which pressure was the only parameter affecting proliferation of cells. Simulations exposed that, due to constant collisions with other growing bacteria, cells have virtually no free space to move sideways, thereby delaying growth and boosting chances of overlapping on top of each other. This scenario was tested experimentally on agar surfaces. Comparison between experiments and simulations suggested that the inside/outside differential pressure determines growth, both timewise and in terms of spatial directions, eventually moulding colony shape. We thus argue that-at least in the case studied-mere physical pressure of growing cells suffices to explain key dynamics of colony formation.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

Despite intensive research on the biochemical and regulatory features of the archetypal catabolic TOL system borne by pWW0 of Pseudomonas putida strain mt-2, the physical arrangement and tridimensional logic of the xyl gene expression flow remains unknown. In this work, the spatial distribution of specific xyl mRNAs with respect to the host nucleoid, the TOL plasmid, and the ribosomal pool has been investigated. In situ hybridization of target transcripts with fluorescent oligonucleotide probes revealed that xyl mRNAs cluster in discrete foci, adjacent but clearly separated from the TOL plasmid and the cell nucleoid. Also, they colocalize with ribosome-rich domains of the intracellular milieu. This arrangement was maintained even when the xyl genes were artificially relocated to different chromosomal locations. The same held true when genes were expressed through a heterologous T7 polymerase-based system, which likewise led to mRNA foci outside the DNA. In contrast, rifampin treatment, known to ease crowding, blurred the confinement of xyl transcripts. This suggested that xyl mRNAs exit from their initiation sites to move to ribosome-rich points for translation-rather than being translated coupled to transcription. Moreover, the results suggest the distinct subcellular motion of xyl mRNAs results from both innate properties of the sequences and the physical forces that keep the ribosomal pool away from the nucleoid in P. putida This scenario is discussed within the background of current knowledge on the three-dimensional organization of the gene expression flow in other bacteria and the environmental lifestyle of this soil microorganism.IMPORTANCE The transfer of information between DNA, RNA, and proteins in a bacterium is often compared to the decoding of a piece of software in a computer. However, the tridimensional layout and the relational logic of the cognate biological hardware, i.e., the nucleoid, the RNA polymerase, and the ribosomes, are habitually taken fo

Boolean NOR gates have been widely implemented in Escherichia coli as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and the metabolic engineering platform Pseudomonas putida with components tailored for E. coli. To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library three inducible promoters that are specific to P. putida, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from E. coli to P. putida, the CELLO software was still able to find an effective NOR gate in the new chassis. The automated generation of the corresponding DNA sequence and in vivo experimental verification accredited that some genetic modules initially optimized for E. coli can indeed be reused to deliver NOR logic in P. putida as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for the quick assembly of genetic circuits to meet complex specifications.

The implementation of Boolean logic circuits in cells have become a very active field within synthetic biology. Although these are mostly focussed on the genetic components alone, the context in which the circuit performs is crucial for its outcome. We characterise 20 genetic NOT logic gates in up to 7 bacterial-based contexts each, to generate 135 different functions. The contexts we focus on are combinations of four plasmid backbones and three hosts, two Escherichia coli and one Pseudomonas putida strains. Each gate shows seven different dynamic behaviours, depending on the context. That is, gates can be fine-tuned by changing only contextual parameters, thus improving the compatibility between gates. Finally, we analyse portability by measuring, scoring, and comparing gate performance across contexts. Rather than being a limitation, we argue that the effect of the genetic background on synthetic constructs expands functionality, and advocate for considering context as a fundamental design parameter.

Genetically encoded logic gates, especially inverters-NOT gates-are the building blocks for designing circuits, engineering biosensors, or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium, and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parametrized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described inverter package (20 different versions of 12 distinct gates) borne by BHR plasmids thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.