Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.

The coordinated control of Ca2+ signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca2+ influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function CNGC mutation (brush) in Lotus japonicus resulting in a leaky tetrameric channel. brush resides in a cluster of redundant CNGCs encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic brush mutation impaired root development and infection by nitrogen-fixing rhizobia. The brush allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the brush phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.

This work presents a new approach to the rapid characterization of antibiotic resistance in environmental settings. For monitoring purposes and risk assessment, it is important to link the detection of resistance genes to the actual carrying species and their potential pathogenicity for humans. Starting from a bacterial culture plate, this proof of principle study developed a colony-based fusion recombinase polymerase amplification (RPA) reaction that detects species and resistance genes simultaneously within 1.5 h. A first step generates the fusion product by homogeneous RPA, while detection occurs in a second step via heterogeneous asymmetric RPA (haRPA) on a flow-based microarray chip. The assay system successfully discriminated between Escherichia coli colonies carrying blaCTX-M cluster 1 resistance genes and E. coli carrying blaCTX-M genes of other clusters as well as other bacterial species carrying blaCTX-M resistance genes. A threshold value of 17% was determined for the differentiation between positive and negative samples. Analysis of water from the river Lech demonstrated the possibility of addressing real environmental samples. The potential for multiplexing was demonstrated by successful formation of fusion products by homogeneous RPA also in Klebsiella pneumoniae. This study uses RPA for the first time to create molecular fusion products providing a promising tool for future multiplex analyses of antibiotic resistance in the environment.

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A method using chemiluminescence-based heterogeneous asymetric recombinase polymerase amplification for the detection and quantification of mycotoxin producers was developed.