We present the construction of layer-by-layer (LbL) assemblies of polyethylenimine and urease onto reduced-graphene-oxide based field-effect transistors (rGO FETs) for the detection of urea. This versatile biosensor platform simultaneously exploits the pH dependency of liquid-gated graphene-based transistors and the change in the local pH produced by the catalyzed hydrolysis of urea. The use of an interdigitated microchannel resulted in transistors displaying low noise, high pH sensitivity (20.3µA/pH) and transconductance values up to 800 µS. The modification of rGO FETs with a weak polyelectrolyte improved the pH response because of its transducing properties by electrostatic gating effects. In the presence of urea, the urease-modified rGO FETs showed a shift in the Dirac point due to the change in the local pH close to the graphene surface. Markedly, these devices operated at very low voltages (less than 500mV) and were able to monitor urea in the range of 1-1000µm, with a limit of detection (LOD) down to 1µm, fast response and good long-term stability. The urea-response of the transistors was enhanced by increasing the number of bilayers due to the increment of the enzyme surface coverage onto the channel. Moreover, quantification of the heavy metal Cu(with a LOD down to 10nM) was performed in aqueous solution by taking advantage of the urease specific inhibition.

Herein, a biosensor based on a reduced graphene oxide field effect transistor (rGO-FET) functionalized with the cascading enzymes arginase and urease was developed for the detection of L-arginine. Arginase and urease were immobilized on the rGO-FET sensing surface via electrostatic layer-by-layer assembly using polyethylenimine (PEI) as cationic building block. The signal transduction mechanism is based on the ability of the cascading enzymes to selectively perform chemical transformations and prompt local pH changes, that are sensitively detected by the rGO-FET. In the presence of L-arginine, the transistors modified with (PEI/urease(arginase)) multilayers showed a shift in the Dirac point due to the change in the local pH close to the graphene surface, produced by the catalyzed urea hydrolysis. The transistors were able to monitor L-arginine in the 10-1000 μM linear range with a LOD of 10 μM, displaying a fast response and a good long-term stability. The sensor showed stereospecificity and high selectivity in the presence of non-target amino acids. Taking into account the label-free, real-time measurement capabilities and the easily quantifiable, electronic output signal, this biosensor offers advantages over state-of-the-art L-arginine detection methods.

Horizontal gene transfer accelerates microbial evolution, promoting diversification and adaptation. The globally abundant marine cyanobacterium Prochlorococcus has a highly streamlined genome with frequent gene exchange reflected in its extensive pangenome. The source of its genomic variability, however, remains elusive since most cells lack the common mechanisms that enable horizontal gene transfer, including conjugation, transformation, plasmids and prophages. Examining 623 genomes, we reveal a diverse system of mobile genetic elements – cargo-carrying transposons we named tycheposons – that shape Prochlorococcus’ genomic plasticity. The excision and integration of tycheposons at seven tRNA genes drive the remodeling of larger genomic islands containing most of Prochlorococcus’ flexible genes. Most tycheposons carry genes important for niche differentiation through nutrient acquisition; others appear similar to phage parasites. Tycheposons are highly enriched in extracellular vesicles and phage particles in ocean samples, suggesting efficient routes for their dispersal, transmission and propagation. Supported by evidence for similar elements in other marine microbes, our work underpins the role of vesicle- and virus-mediated transfer of mobile genetic elements in the diversification and adaptation of microbes in dilute aquatic environments – adding a significant piece to the puzzle of what governs microbial evolution in the planet’s largest habitat.

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As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.