Abstract Cell-free expression systems enable rapid prototyping of genetic programs in vitro. However, current throughput of cell-free measurements is limited by the use of channel-limited fluorescent readouts. Here, we describe DNA Regulatory element Analysis by cell-Free Transcription and Sequencing (DRAFTS), a rapid and robust in vitro approach for multiplexed measurement of transcriptional activities from thousands of regulatory sequences in a single reaction. We employ this method in active cell lysates developed from ten diverse bacterial species. Interspecies analysis of transcriptional profiles from > 1,000 diverse regulatory sequences reveals functional differences in promoter activity that can be quantitatively modeled, providing a rich resource for tuning gene expression in diverse bacterial species. Finally, we examine the transcriptional capacities of dual-species hybrid lysates that can simultaneously harness gene expression properties of multiple organisms. We expect that this cell-free multiplex transcriptional measurement approach will improve genetic part prototyping in new bacterial chassis for synthetic biology.

Recent efforts in synthetic biology have shown the possibility of engineering distributed functions in populations of living cells, which requires the development of highly orthogonal, genetically encoded communication pathways. Cell-free transcription-translation (TXTL) reactions encapsulated in microcompartments enable prototyping of molecular communication channels and their integration into engineered genetic circuits by mimicking critical cell features, such as gene expression, cell size, and cell individuality within a community. In this review, we discuss the uses of cell-free transcription–translation reactions for the development of synthetic genetic circuits, with a special focus on the use of microcompartments supporting this reaction. We highlight several studies where molecular communication between non-living microcompartments and living cells have been successfully engineered.

Detarioideae is well known for its high diversity of floral traits, including flower symmetry, number of organs, and petal size and morphology. This diversity has been characterized and studied at higher taxonomic levels, but limited analyses have been performed among closely related genera with contrasting floral traits due to the lack of fully resolved phylogenetic relationships. Here, we used four representative transcriptomes to develop an exome capture (target enrichment) bait for the entire subfamily and applied it to the Anthonotha clade using a complete data set (61 specimens) representing all extant floral diversity. Our phylogenetic analyses recovered congruent topologies using ML and Bayesian methods. Anthonotha was recovered as monophyletic contrary to the remaining three genera (Englerodendron, Isomacrolobium and Pseudomacrolobium), which form a monophyletic group sister to Anthonotha. We inferred a total of 35 transitions for the seven floral traits (pertaining to flower symmetry, petals, stamens and staminodes) that we analyzed, suggesting that at least 30% of the species in this group display transitions from the ancestral condition reconstructed for the Anthonotha clade. The main transitions were towards a reduction in the number of organs (petals, stamens and staminodes). Despite the high number of transitions, our analyses indicate that the seven characters are evolving independently in these lineages. Petal morphology is the most labile floral trait with a total of seven independent transitions in number and seven independent transitions to modification in petal types. The diverse petal morphology along the dorsoventral axis of symmetry within the flower is not associated with differences at the micromorphology of petal surface, suggesting that in this group all petals within the flower might possess the same petal identity at the molecular level. Our results provide a solid evolutionary framework for further detailed analyses of the molecular basis of petal identity.

The bottom-up assembly of synthetic cell systems capable of recapitulating biological functions has become a means to understand living matter by construction. The integration of biomolecular components into active, cell-sized, genetically programmed compartments remains, however, a major bottleneck for building synthetic cells. A primary feature of real cells is their ability to actively interact with their surroundings, particularly in stressed conditions. Here, we construct a synthetic cell equipped with an inducible genetic circuit that responds to changes in osmotic pressure through the mechanosensitive channel MscL. Liposomes loaded with an E. coli cell-free transcription-translation (TXTL) system are induced with IPTG when exposed to hypo-osmotic solution, resulting in the expression of a bacterial cytoskeletal protein (MreB). MreB associates with the membrane to generate a cortex-like structure. Our work provides the first example of molecular integration that couples mechanosensitivity, gene expression, and self-assembly at the inner membrane of synthetic cells.

Known for its remarkable biodiversity and high levels of endemism, the Brazilian Atlantic Rainforest has been characterized as one of the most threatened biomes on the planet. Despite strong interest in recent years, we still lack a comprehensive scenario to explain the origin and maintenance of diversity in this region, partially given the relatively low power of analyses involving few independent genetic loci. In this study, we examine a phylogenomic dataset of five ant species to investigate phylogeographical patterns across the Brazilian Atlantic Forest. We sequenced ultraconserved elements to generate hundreds of loci using a bait set developed specifically for hymenopterans. We analyzed the data using Bayesian and maximum likelihood approaches of phylogenetic inference. Results were then integrated with environmental niche modeling of current and past climates, including the Last Glacial Maximum and the last interglacial period. The studied species showed differentiation patterns that were consistent with the north/south division of the Atlantic Rainforest indicated in previous studies for other taxa. However, there were differences among species, both in the location of phylogeographic breaks and in the pattern of genetic variation within these areas. Samples from southern localities tended to show recent genetic structure, although a site in Tapiraí (state of São Paulo) repeatedly showed an intriguing older history of differentiation. All species experienced shifts in areas of suitability through the time. Our study suggests that distinct groups may have responded idiosyncratically to the climatic changes that took place in the Brazilian Atlantic Forest. The amount of intraspecific genetic structure was related to the inferred geographical distribution of habitat suitability according to current and past times. Also, a parallel between the amount of Quaternary climatic suitability and the level of interspecific differentiation was detected for four species. Finally, despite strong contractions at the northeastern region of the forest, the remaining areas appear to have been able to act as refugia.

The sunflower family, Asteraceae, comprises 10% of all flowering plant species and displays an incredible diversity of form. Asteraceae are clearly monophyletic, yet resolving phylogenetic relationships within the family has proven difficult, hindering our ability to understand its origin and diversification. Recent molecular clock dating has suggested a Cretaceous origin, but the lack of deep sampling of many genes and representative taxa from across the family has impeded the resolution of migration routes and diversifications that led to its global distribution and tremendous diversity. Here we use genomic data from 256 terminals to estimate evolutionary relationships, timing of diversification(s), and biogeographic patterns. Our study places the origin of Asteraceae at ∼83 MYA in the late Cretaceous and reveals that the family underwent a series of explosive radiations during the Eocene which were accompanied by accelerations in diversification rates. The lineages that gave rise to nearly 95% of extant species originated and began diversifying during the middle Eocene, coincident with the ensuing marked cooling during this period. Phylogenetic and biogeographic analyses support a South American origin of the family with subsequent dispersals into North America and then to Asia and Africa, later followed by multiple worldwide dispersals in many directions. The rapid mid-Eocene diversification is aligned with the biogeographic range shift to Africa where many of the modern-day tribes appear to have originated. Our robust phylogeny provides a framework for future studies aimed at understanding the role of the macroevolutionary patterns and processes that generated the enormous species diversity of Asteraceae.

Development of oligonucleotide probes facilitates chromosome identification via fluorescence in situ hybridization (FISH) in many organisms.

CRISPR-Cas systems inherently multiplex through CRISPR arrays—whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis., CRISPR array generation is difficult due to reoccurring repeat sequences. Here the authors present CRATES—a modular, one-pot assembly method—and demonstrate the creation of arrays for Cas9, Cas12a and Cas13a for cell-free, bacterial, yeast and mammalian systems.