Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels.

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Water potential governs the effector specificity of the transcriptional regulator XylR of Pseudomonas putida.

The biodegradative capacity of bacteria in their natural habitats is affected by water availability. In this work, we have examined the activity and effector specificity of the transcriptional regulator XylR of the TOL plasmid pWW0 of Pseudomonas putida mt-2 for biodegradation of m-xylene when external water potential was manipulated with polyethylene glycol PEG8000. By using non-disruptive luxCDEAB reporter technology, we noticed that the promoter activated by XylR (Pu) restricted its activity and the regulator became more effector-specific towards head TOL substrates when cells were grown under water subsaturation. Such a tight specificity brought about by water limitation was relaxed when intracellular osmotic stress was counteracted by the external addition of the compatible solute glycine betaine. With these facts in hand, XylR variants isolated earlier as effector-specificity responders to the non-substrate 1,2,4-trichlorobenzene under high matric stress were re-examined and found to be unaffected by water potential in vivo. All these phenomena could be ultimately explained as the result of water potential-dependent conformational changes in the A domain of XylR and its effector-binding pocket, as suggested by AlphaFold prediction of protein structures. The consequences of this scenario for the evolution of specificities in regulators and the emergence of catabolic pathways are discussed.

Key reaction components affect the kinetics and performance robustness of cell-free protein synthesis reactions.

Cell-free protein synthesis (CFPS) reactions have grown in popularity with particular interest in applications such as gene construct prototyping, biosensor technologies and the production of proteins with novel chemistry. Work has frequently focussed on optimising CFPS protocols for improving protein yield, reducing cost, or developing streamlined production protocols. Here we describe a statistical Design of Experiments analysis of 20 components of a popular CFPS reaction buffer. We simultaneously identify factors and factor interactions that impact on protein yield, rate of reaction, lag time and reaction longevity. This systematic experimental approach enables the creation of a statistical model capturing multiple behaviours of CFPS reactions in response to components and their interactions. We show that a novel reaction buffer outperforms the reference reaction by 400% and importantly reduces failures in CFPS across batches of cell lysates, strains of E. coli, and in the synthesis of different proteins. Detailed and quantitative understanding of how reaction components affect kinetic responses and robustness is imperative for future deployment of cell-free technologies.

Cell-free protein synthesis in hydrogel materials.

We report a method for embedding cell-free protein synthesis reactions in macro-scale hydrogel materials without a free liquid phase. This paper focuses on methods of preparation for a variety of hydrogels and an investigation of the impact that the hydrogel material has on cell-free protein synthesis.

Bovistol B, bovistol D and strossmayerin: Sesquiterpene metabolites from the culture filtrate of the basidiomycete Coprinopsis strossmayeri.

Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B (1) and D (2), and one monomeric sesquiterpene, strossmayerin (7), were isolated from the culture filtrate of the basidiomycete fungus Coprinopsis strossmayeri. The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1-11 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2.

Genome Sequence of Lecanicillium fungicola 150-1, the Causal Agent of Dry Bubble Disease.

The fungus Lecanicillium fungicola causes dry bubble disease in the white button mushroom Agaricus bisporus Control strategies are limited, as both the host and pathogen are fungi, and there is limited understanding of the interactions in this pathosystem. Here, we present the genome sequence of Lecanicillium fungicola strain 150-1.

Draft Genome Sequence of the Coprinoid Mushroom Coprinopsis strossmayeri.

Coprinopsis strossmayeri is a coprinoid mushroom favoring the habitat of herbivore dung. As a result of this highly competitive environment, C. strossmayeri is anticipated to produce a wide array of antimicrobial secondary metabolites (SMs) of potential pharmaceutical importance. Here, we present the draft genome sequence of C. strossmayeri.