Feedback mechanisms play a critical role in the maintenance of cell homeostasis in the presence of disturbances and uncertainties. Motivated by the need to tune the dynamics and improve the robustness of synthetic gene circuits, biological engineers have proposed various designs that mimic natural molecular feedback control mechanisms. However, practical and predictable implementations have proved challenging because of the complexity of synthesis and analysis of complex biomolecular networks. Here, we analyze and experimentally validate a first synthetic biomolecular controller executed in vitro. The controller is based on the interaction between a sigma and an anti-sigma factor, which ensures that gene expression tracks an externally imposed reference level, and achieves this goal even in the presence of disturbances. Our design relies upon an analog of the well-known principle of integral feedback in control theory. We implement the controller in an Escherichia coli cell-free transcription-translation (TXTL) system, a platform that allows rapid prototyping and implementation. Modeling and theory guide experimental implementation of the controller with well-defined operational predictability.

Tree species in the genus Cedrela P. Browne are threatened by timber overexploitation across the Neotropics. Genetic identification of processed timber can be used to supplement wood anatomy to assist in the taxonomic and source validation of protected species and populations of Cedrela. However, few genetic resources exist that enable both species and source identification of Cedrela timber products. We developed several ‘omic resources including a leaf transcriptome, organelle genome (cpDNA), and diagnostic single nucleotide polymorphisms (SNPs) that may assist the classification of Cedrela specimens to species and geographic origin and enable future research on this widespread Neotropical tree genus.

Whole-chromosome painting probes were developed for each of the 10 chromosomes of maize by producing amplifiable libraries of unique sequences of oligonucleotides that can generate labeled probes through transcription reactions. These paints allow identification of individual homologous chromosomes for many applications as demonstrated in somatic root tip metaphase cells, in the pachytene stage of meiosis, and in interphase nuclei. Several chromosomal aberrations were examined as proof of concept for study of various rearrangements using probes that cover the entire chromosome and that label diverse varieties. The relationship of the supernumerary B chromosome and the normal chromosomes was examined with the finding that there is no detectable homology between any of the normal A chromosomes and the B chromosome. Combined with other chromosome-labeling techniques, a complete set of whole-chromosome oligonucleotide paints lays the foundation for future studies of the structure, organization, and evolution of genomes.

During periods of reduced O2 supply, the most profound changes in gene expression are mediated by hypoxia-inducible factor (HIF) transcription factors that play a key role in cellular responses to low-O2 tension. Using target-enrichment sequencing, we tested whether variation in 26 genes in the HIF signaling pathway was associated with high altitude and therefore corresponding O2 availability in three duck species that colonized the Andes from ancestral low-altitude habitats in South America. We found strong support for convergent evolution in the case of two of the three duck species with the same genes (EGLN1, EPAS1), and even the same exons (exon 12, EPAS1), exhibiting extreme outliers with a high probability of directional selection in the high-altitude populations. These results mirror patterns of adaptation seen in human populations, which showed mutations in EPAS1, and transcriptional regulation differences in EGLN1, causing changes in downstream target transactivation, associated with a blunted hypoxic response.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immune defense systems, which are widely distributed in bacteria and Archaea, can provide sequence-specific protection against foreign DNA or RNA in some cases. However, the evolution of defense systems in bacterial hosts did not lead to the elimination of phages, and some phages carry anti-CRISPR genes that encode products that bind to the components mediating the defense mechanism and thus antagonize CRISPR-Cas immune systems of bacteria. Given the extensive application of CRISPR-Cas9 technologies in gene editing, in this review, we focus on the anti-CRISPR proteins (Acrs) that inhibit CRISPR-Cas systems for gene editing. We describe the discovery of Acrs in immune systems involving type I, II, and V CRISPR-Cas immunity, discuss the potential function of Acrs in inactivating type II and V CRISPR-Cas systems for gene editing and gene modulation, and provide an outlook on the development of important biotechnology tools for genetic engineering using Acrs.

Abstract. Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing t

Rationale Cell-free transcription-translation (TXTL) is becoming a popular technology to prototype and engineer biological systems outside living organisms. TXTL relies commonly to a cytoplasmic extract that provides the molecular components necessary to recapitulate gene expression in vitro, where most of the available systems are derived from E. coli. The proteinic and enzymatic composition of lysates, however, is typically unknown. In this work, we analyzed by mass spectrometry the molecular constituents of the all-E. coli TXTL platform myTXTL prepared from the E. coli strain BL21 Rosetta2. Methods Standard TXTL reactions were assembled and executed for 10-12 hours at 29°C. In addition to a no-DNA control, four DNA programs were executed in separate reactions to synthesize the reporter protein deGFP as well as the phages MS2, phix174 and T7. The reactions were treated according to standard procedures (trypsin treatment, cleaning) before performing liquid chromatography-mass spectrometry (LC-MS). Data analysis was performed using Sequest and protein identification using Scaffold. Results 500-800 proteins were identified by LC-MS in the blank reactions. We organized the most abundant protein sets into several categories pertaining, in particular, to transcription, translation and ATP regeneration. The synthesis of deGFP was easily measured. The major structural proteins that compose the three phages MS2, phix174 and T7 were also identified. Conclusions Mass spectrometry is a practical tool to characterize biochemical solutions as complex as a cell-free TXTL reaction and to determine the presence of synthesized proteins. The data presented demonstrate that the composition of TXTL based on lysates can be used to validate some underlying molecular mechanisms implicated in cell-free protein synthesis. The composition of the lysate shows significant differences with respect to similar studies on other E. coli strains.

Synthesizing proteins inside liposomes and other micro-compartments is today a well-established practice. However, the origin of this research is not distant in time, dating back to the 1999-2004 period, where the first successful attempts were published. Protein synthesis inside artificial compartments is now under strong expansion in the context of synthetic biology (in the “bottom up” approaches), and in particular it strongly contributes to the construction of artificial cell-like systems. These systems, often called “synthetic cells”, can be used to model cellular processes, including membrane-centred ones. They are very innovative models that complement traditional studies and promise future applications. This review does not discuss all current directions in synthetic cell research; in particular it does not include all kind of artificial compartments. Instead, it is uniquely dedicated to the analysis of historical and technical developments of protein synthesis inside liposomes, highlighting a selected list of open questions. One of the goals is remarking the importance of mastering liposome technology together to cell-free systems for the successful realization of this specific type of synthetic cells. At this aim, four currently employed protocols are compared and commented, with major emphasis on the droplet transfer method, which is attractive due to its simplicity and encapsulation efficiency.