Operon Research Articles

Modular Cloning (MoClo) toolkits enable the rapid assembly of multigene constructs. They are based on Golden Gate cloning, which uses Type IIS restriction enzymes that cut outside their recognition site. Since recognition and cut sequence are decoupled, overhangs created by Type IIS restriction enzymes can be deliberately chosen, and cloning strategies typically prevent cutting of correctly joined DNA fragments. This allows highly efficient assemblies of multiple DNA fragments in a single reaction. In MoClo, individual functional DNA parts such as promoters, ribosomal binding sites, coding sequences, and terminators, as well as higher-order assemblies, are assigned standardized overhangs, such that reusable parts libraries can be created. The majority of bacterial MoClo toolkits are designed for cloning monocistronic transcriptional units and do not provide a structured path for the assembly of polycistronic operons. This protocol demonstrates the assembly of polycistronic transcription units with the In- & Out-Cloning toolkit. The same approach is transferable to other MoClo toolkits.

The main purpose of this study is to examine the spatial impact of Türkiye's cross-border operations on Iraq, particularly focusing on their potential to destabilize the northern region using Geographical Information Systems (GIS). Türkiye's operations are often criticized in both political discourse and academic literature, with the argument that they disrupt the stability of Iraq's northern provinces, which are considered to be among the most stable parts of the country. To investigate this claim, this study employs Global Moran's I* and Getis-Ord General G analyses to assess the spatial distribution of attack intensity across Iraqi provinces. The time period considered is the post-occupation era. To further delve into the dynamics of violence, the analysis will be divided into three sub-periods based on significant changes in attack patterns in general: 2004-2012, 2013-2017, and 2018-2023. Additionally, to ensure the reliability of the results, the analyses will be conducted both with and without data on Türkiye's involvement. To enhance the robustness of the findings, comparative analyses will be conducted both with and without incorporating data on Türkiye's military interventions. The results of this study will provide valuable insights into the complex relationship between cross-border operations and regional stability in the Middle East. The study concluded that Türkiye's operations did not statistically affect the spatial distribution of violence in North of Iraq.

Bacterial biosynthetic gene clusters (BGCs) drive the production of diverse bioactive specialized metabolites regulated by transcription factors (TFs) in response to environmental signals. In this meta-analysis, we investigated the regulatory architecture of BGCs by compiling experimental data sets of transcription factor binding sites (TFBSs) to unveil (i) the functional gene categories preferentially targeted by TFs, (ii) the transcriptional coverage based on cluster organization, (iii) the positional distribution of TFBSs, and (iv) the binding strength of TFs. Our analysis reveals a strategy where global TFs primarily target monocistronic pathway-specific TFs when present, aligning with a "one-for-all" strategy ensuring cluster-wide expression control. In contrast, biosynthetic genes are typically organized into polycistronic operons, promoting the synchronized production of building blocks, core structures, and modifications. Assessment of the TF-TFBS interaction strength indicates that TFBSs within BGCs exhibit lower binding affinities compared to those associated with core regulon genes outside BGCs. This lower binding affinity allows greater regulatory flexibility, as BGC expression often responds to multiple environmental signals, each one sensed by its specific TF. These findings refine our understanding of the regulatory logic shaping BGC expression and offer valuable insights for predicting activation conditions, facilitating the discovery of novel compounds through targeted culture and engineering strategies.

Repurposing natural systems to develop customized functions in biological systems is one of the main thrusts of synthetic biology. Translational coupling is a common phenomenon in diverse polycistronic operons for efficient allocation of limited genetic space and cellular resources. These beneficial features of translation coupling can provide exciting opportunities for creating novel synthetic biological devices. Here, we introduce a modular synthetic translational coupling element (synTCE) and integrate this design with de novo designed riboregulators,toehold switches. A systematic exploration of sequence domain variants for synTCEs led to the identification of critical design considerations for improving the system performance. Next, this design approach was seamlessly integrated into logic computations and applied to construct multi-output transcripts with well-defined stoichiometric control. This module was further applied to signaling cascades for combined signal transduction and multi-input/multi-output synthetic devices. Further, the synTCEs can precisely manipulate the N-terminal ends of output proteins, facilitating effective protein localization and cellular population control. Therefore, the synTCEs could enhance computational capability and applicability of riboregulators for reprogramming biological systems, leading to future applications in synthetic biology, metabolic engineering and biotechnology.

Vital organismal processes, including development, differentiation and adaptation, involve altered gene expression. Although expression is frequently controlled at the transcriptional stage, various regulation mechanisms operate at downstream levels. Here, we leverage the photoreceptor NmPAL to optogenetically induce RNA refolding and the translation of bacterial mRNAs. Blue-light-triggered NmPAL binding disrupts a cis-repressed mRNA state, thereby relieves obstruction of translation initiation, and upregulates gene expression. Iterative probing and optimization of the circuit, dubbed riboptoregulator, enhanced induction to 30-fold. Given action at the mRNA level, the riboptoregulator can differentially regulate individual structural genes within polycistronic operons. Moreover, it is orthogonal to and can be wed with other gene-regulatory circuits for nuanced and more stringent gene-expression control. We thus advance the pAurora2 circuit that combines transcriptional and translational mechanisms to optogenetically increase bacterial gene expression by >1000-fold. The riboptoregulator strategy stands to upgrade numerous regulatory circuits and widely applies to expression control in microbial biotechnology, synthetic biology and materials science.

Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

Cross-evaluation of E. coli’s operon structures via a whole-cell model suggests alternative cellular benefits for low- versus high-expressing operons

The exploitation of many deposits in Armenia is carried out mainly in an open-pit manner. The development of an open-pit mineral deposit has a negative impact on the atmospheric air due to the result of dust and gas formation. The main sources of harmful emissions during the open-pit mining of mineral deposits are drilling and blasting, loading and transportation operations, and rock dumping. Moreover, drilling and blasting operations account for about 40 % of the total mass of pollutants that harm the environment During the development of mineral deposits occurs a powerful man-made impact on the ecology of the environment from resulting fine dust, which is released during drilling and blasting operations. Significant amounts of dust migrate long distances from the quarry. When downhole charges of explosives (explosives) explode, a shock wave is formed around the charging chamber under the influence of a detonation wave. Under the action of a shock wave, the rock around the well is re-crushed into fine dust, which is ejected through the bottom hole parts into the open surface of the ledge. Under the influence of detonation products of downhole explosive charges, an air shock wave is formed on the surface of the ledge, under the action of which the over-ground fine dust is released into the atmosphere. The growth of mining is exacerbating the processes of climate change and environmental pollution. The task of choosing the method is to increase the efficiency of dust suppression and increase the efficiency of explosive energy in mass explosions at quarries In this regard, the development of ways to reduce the concentration of dust and gas emissions and increase the efficiency of dust suppression released during mass explosions in deep quarries is an urgent environmental task.

Bacterial small RNAs (sRNAs) function in post-transcriptional regulatory responses to environmental changes. However, the lack of eukaryotic RNA interference-like machinery in bacteria has limited the systematic engineering of RNA repression. Here, we report the development of clustered regularly interspaced short palindromic repeats (CRISPR)-guided dead CRIPSR-associated protein 13a (dCas13a) ribonucleoprotein that utilizes programmable CRISPR RNAs (crRNAs) to repress trans-acting and cis-acting sRNA as the target, altering regulatory mechanisms and stress-related phenotypes. In addition, we implemented a modular loop engineering of the crRNA to promote modular repression of the target gene with 92% knockdown efficiency and a single base-pair mismatch specificity. With the engineered crRNAs, we achieved targetable single-gene repression in the polycistronic operon. For metabolic application, 102 crRNAs were constructed in the biofoundry and used for screening novel knockdown sRNA targets to improve lycopene (colored antioxidant) production in Escherichia coli. The CRISPR-dCas13a system will assist as a valuable systematic tool for the discovery of novel sRNAs and the fine-tuning of bacterial RNA repression in both scientific and industrial applications.

ПОБИТОВЫЕ ГОМОМОРФНЫЕ ОПЕРАЦИИ НАД ЧИСЛАМИ С ПЛАВАЮЩЕЙ ТОЧКОЙ

Selective RNA processing and stabilization (SRPS) facilitates the differential expression of multiple genes in polycistronic operons. However, how the coordinated actions of SRPS-related enzymes affect stoichiometric regulation remains unclear. In the present study, the first genome-wide targetome analysis is reported of these enzymes in Escherichia coli, at a single-nucleotide resolution. A strictly linear relationship is observed between the RNA pyrophosphohydrolase processing ratio and scores assigned to the first three nucleotides of the primary transcript. Stem-loops associated with PNPase targetomes exhibit a folding free energy that is negatively correlated with the termination ratio of PNPase at the 3' end. More than one-tenth of the RNase E processing sites in the 5'-untranslated regions（UTR） form different stem-loops that affect ribosome-binding and translation efficiency. The effectiveness of the SRPS elements is validated using a dual-fluorescence reporter system. The findings highlight a multi-layer and quantitative regulatory method for optimizing the stoichiometric expression of genes in bacteria and promoting the application of SRPS in synthetic biology.

Methane is a potent greenhouse gas, which has contributed to approximately a fifth of global warming since pre-industrial times. The agricultural sector produces significant methane emissions, especially from livestock, waste management and rice cultivation. Rice fields alone generate around 9% of total anthropogenic emissions. Methane is produced in waterlogged paddy fields by methanogenic archaea, and transported to the atmosphere through the aerenchyma tissue of rice plants. Thus, bioengineering rice with catalysts to detoxify methane en route could contribute to an efficient emission mitigation strategy. Particulate methane monooxygenase (pMMO) is the predominant methane catalyst found in nature, and is an enzyme complex expressed by methanotrophic bacteria. Recombinant expression of pMMO has been challenging, potentially due to its membrane localization, multimeric structure, and polycistronic operon. Here we show the first steps towards the engineering of plants for methane detoxification with the three pMMO subunits expressed in the model systems tobacco and Arabidopsis. Membrane topology and protein–protein interactions were consistent with correct folding and assembly of the pMMO subunits on the plant ER. Moreover, a synthetic self-cleaving polypeptide resulted in simultaneous expression of all three subunits, although low expression levels precluded more detailed structural investigation. The work presents plant cells as a novel heterologous system for pMMO allowing for protein expression and modification.

The enteropathogen Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) utilizes a cluster of genes encoded within the pathogenicity island 2 (SPI-2) of its genome to proliferate inside macrophages. The expression of SPI-2 is controlled by a complex network of transcriptional regulators and environmental cues, which now include a recently characterized DNA-binding protein named PagR. Growth of S. Typhimurium in low-phosphate, low-magnesium medium mimics conditions inside macrophages. Under such conditions, PagR ensures SPI-2 induction by upregulating the transcription of slyA, which encodes a known activator of SPI-2. Here, we report that PagR represses the expression of a divergently transcribed polycistronic operon that encodes the two subunits of transketolase TktC (i.e., tktD, tktE) of this bacterium. Transketolases contribute to the nonredox rearrangements of phosphorylated sugars of the pentose phosphate pathway, which provide building blocks for amino acids, nucleotides, cofactors, etc. We also demonstrate that PagR represses the expression of its own gene and define two PagR-binding sites between stm2344 and pagR.

COLLECTIONS AND THE METHODOLOGY OF PERFORMING OPERATIONS ON THEM

The operations in spectral fuzzy graphs such as Cartesian product, strong product, bipartite double, Kronecker product and Lexicographic product (composition) are analysed to elucidate the impact of eigenvalue spectra. Also, the properties are derived which delves the effect on spectral fuzzy graphs.

Clostridium tyrobutyricum has great potential for bio-based chemicals and biofuel production from mannitol; however, the mannitol metabolic pathway and its metabolic regulatory mechanism have not been elucidated. To this end, the RNA-seq analysis on the mid-log growth phase of C. tyrobutyricum grown on mannitol or xylose was performed. Comparative transcriptome analysis and co-transcription experiment indicated that mtlARFD, which encodes the mannitol-specific IIA component, transcription activator, mannitol-specific IIBC components, and mannitol-1-phosphate 5-dehydrogenase, respectively, formed a polycistronic operon and could be responsible for mannitol uptake and metabolism. In addition, comparative genomic analysis of the mtlARFD organization and the MtlR protein structural domain among various Firmicutes strains identified the putative cre (catabolite-responsive element) sites and conserved phosphorylation sites, but whether the expression of mannitol operon was affected by CcpA- and MtlR-mediated metabolic regulation during mixed substrate fermentation needs to be further verified experimentally. Based on the gene knockout and complementation results, the predicted mannitol operon mtlARFD was confirmed to be responsible for mannitol utilization in C. tyrobutyricum. The results of this study could be used to enhance the mannitol metabolic pathway and explore the potential metabolic regulation mechanism of mannitol during mixed substrate fermentation.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3′-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5′-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

The rational design and realisation of simple-to-use genetic control elements that are modular, orthogonal and robust is essential to the construction of predictable and reliable biological systems of increasing complexity. To this effect, we introduce modular Artificial RNA interference (mARi), a rational, modular and extensible design framework that enables robust, portable and multiplexed post-transcriptional regulation of gene expression in Escherichia coli. The regulatory function of mARi was characterised in a range of relevant genetic contexts, demonstrating its independence from other genetic control elements and the gene of interest, and providing new insight into the design rules of RNA based regulation in E. coli, while a range of cellular contexts also demonstrated it to be independent of growth-phase and strain type. Importantly, the extensibility and orthogonality of mARi enables the simultaneous post-transcriptional regulation of multi-gene systems as both single-gene cassettes and poly-cistronic operons. To facilitate adoption, mARi was designed to be directly integrated into the modular BASIC DNA assembly framework. We anticipate that mARi-based genetic control within an extensible DNA assembly framework will facilitate metabolic engineering, layered genetic control, and advanced genetic circuit applications.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3’-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.

In bacteria, small non-coding RNAs (sRNAs) bind to target mRNAs and regulate their translation and/or stability. In the polycistronic galETKM operon of Escherichia coli, binding of the Spot 42 sRNA to the operon transcript leads to the generation of galET mRNA. The mechanism of this regulation has remained unclear. We show that sRNA-mRNA base pairing at the beginning of the galK gene leads to both transcription termination and transcript cleavage within galK, and generates galET mRNAs with two different 3'-OH ends. Transcription termination requires Rho, and transcript cleavage requires the endonuclease RNase E. The sRNA-mRNA base-paired segments required for generating the two galET species are different, indicating different sequence requirements for the two events. The use of two targets in an mRNA, each of which causes a different outcome, appears to be a novel mode of action for a sRNA. Considering the prevalence of potential sRNA targets at cistron junctions, the generation of new mRNA species by the mechanisms reported here might be a widespread mode of bacterial gene regulation.