Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze–thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive β-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.
Temnothorax (Formicidae: Myrmicinae) is a diverse genus of ants found in a broad spectrum of ecosystems across the northern hemisphere. These diminutive ants have long served as models for social insect behavior, leading to discoveries about social learning and inspiring hypotheses about the process of speciation and the evolution of social parasitism. This genus is highly morphologically and behaviorally diverse, and this has caused a great deal of taxonomic confusion in recent years. Past efforts to estimate the phylogeny of this genus have been limited in taxonomic scope, leaving the broader evolutionary patterns in Temnothorax unclear. To establish the monophyly of Temnothorax, resolve the evolutionary relationships, reconstruct the historical biogeography and investigate trends in the evolution of key traits, I generated, assembled, and analyzed two molecular datasets: a traditional multi-locus Sanger sequencing dataset, and an ultra-conserved element (UCE) dataset. Using maximum likelihood, Bayesian, and summary-coalescent based approaches, I analyzed 22 data subsets consisting of 103 ingroup taxa and a maximum of 1.8 million base pairs in 2485 loci.
Although anecdotally associated with local bears (Ursus arctos and U. thibetanus), the exact identity of ‘hominid’-like creatures important to folklore and mythology in the Tibetan Plateau–Himalaya region is still surrounded by mystery. Recently, two purported yeti samples from the Himalayas showed genetic affinity with an ancient polar bear, suggesting they may be from previously unrecognized, possibly hybrid, bear species, but this preliminary finding has been under question. We conducted a comprehensive genetic survey of field-collected and museum specimens to explore their identity and ultimately infer the evolutionary history of bears in the region. Phylogenetic analyses of mitochondrial DNA sequences determined clade affinities of the purported yeti samples in this study, strongly supporting the biological basis of the yeti legend to be local, extant bears. Complete mitochondrial genomes were assembled for Himalayan brown bear (U. a. isabellinus) and black bear (U. t. laniger) for the first time. Our results demonstrate that the Himalayan brown bear is one of the first-branching clades within the brown bear lineage, while Tibetan brown bears diverged much later. The estimated times of divergence of the Tibetan Plateau and Himalayan bear lineages overlap with Middle to Late Pleistocene glaciation events, suggesting that extant bears in the region are likely descendants of populations that survived in local refugia during the Pleistocene glaciations.
Species identification using DNA sequences, known as DNA barcoding has been widely used in many applied fields. Current barcoding methods are usually based on a single mitochondrial locus, such as cytochrome c oxidase subunit I (COI). This type of barcoding method does not always work when applied to species separated by short divergence times or that contain introgressed genes from closely related species. Herein we introduce a more effective multi-locus barcoding framework that is based on gene capture and “next-generation” sequencing. We selected 500 independent nuclear markers for ray-finned fishes and designed a three-step pipeline for multilocus DNA barcoding. We applied our method on two exemplar datasets each containing a pair of sister fish species: Siniperca chuatsi vs. Sini. kneri and Sicydium altum vs. Sicy. adelum, where the COI barcoding approach failed. Both of our empirical and simulated results demonstrated that under limited gene flow and enough separation time, we could correctly identify species using multilocus barcoding method. We anticipate that, as the cost of DNA sequencing continues to fall that our multilocus barcoding approach will eclipse existing single-locus DNA barcoding methods as a means to better understand the diversity of the living world.
The extinct stilt-legged equids of North America are not related to Asiatic asses or horses, but instead represent a distinct lineage outside of living equid diversity that became extinct in the terminal Pleistocene.
Weevils (Curculionoidea) comprise one of the most diverse groups of organisms on earth. There is hardly a vascular plant or plant part without its own species of weevil feeding on it and weevil species diversity is greater than the number of fishes, birds, reptiles, amphibians and mammals combined. Here, we employ ultraconserved elements (UCEs) designed for beetles and a novel partitioning strategy of loci to help resolve phylogenetic relationships within the radiation of Australasian smurf-weevils (Eupholini). Despite being emblematic of the New Guinea fauna, no previous phylogenetic studies have been conducted on the Eupholini. In addition to a comprehensive collection of fresh specimens, we supplement our taxon sampling with museum specimens, and this study is the first target enrichment phylogenomic dataset incorporating beetle specimens from museum collections. We use both concatenated and species tree analyses to examine the relationships and taxonomy of this group. For species tree analyses we present a novel partitioning strategy to better model the molecular evolutionary process in UCEs. We found that the current taxonomy is problematic, largely grouping species on the basis of similar color patterns. Finally, our results show that most loci required multiple partitions for nucleotide rate substitution, suggesting that single partitions may not be the optimal partitioning strategy to accommodate rate heterogeneity for UCE loci.
Premise of the study: Seed dispersal contributes to gene flow and is responsible for colonization of new sites and range expansion. Sequencing chloroplast haplotypes offers a way to estimate contributions of seed dispersal to population genetic structure and enables studies of population history. Whole-genome sequencing is expensive, but resources can be conserved by pooling samples. Unfortunately, haplotype associations among single-nucleotide polymorphisms (SNPs) are lost in pooled samples, and treating SNP allele frequencies as independent markers provides biased estimates of genetic structure. Methods: We developed sampling methodologies and an application, CallHap, that uses a least-squares algorithm to evaluate the fit between observed and predicted SNP allele frequencies from pooled samples based on haplotype network phylogeny structure, thus enabling pooling for chloroplast sequencing for large-scale studies of chloroplast genomic variation. This method was tested using artificially constructed test networks and pools, and pooled samples of Lasthenia californica (California goldfields) from southern Oregon, USA. Results: CallHap reliably recovered network topologies and haplotype frequencies from pooled samples. Discussion: The CallHap pipeline allows for the efficient use of resources for estimation of genetic structure for studies using nonrecombining haplotypes such as intraspecific variation in chloroplast, mitochondrial, bacterial, or viral DNA.
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