Synthetic Elements Research Articles

Exploring cyanobacteria seed resources and corresponding gas-vesicle-biosynthetic elements for producing ultrasound molecular imaging probes

Use of gas vesicles (GVs) as biosynthetic ultrasound molecular imaging (UMI) probes has become a hot topic of research in recent years owing to their advantages of high stability, low risk of toxicity, genetic engineering characterization, easy post-modification, and drug-loading potential. Exploration of natural GV biosynthetic resources and their corresponding biosynthetic elements for various ultrasonic diagnosis and treatment needs to be developed in this field. In this study, we utilized the sequenced cyanobacteria resources preserved in the Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB) to screen seed cells and corresponding synthetic elements for biosynthesis of UMI probes. GV operons from the genomes of 158 strains of cyanobacteria were analyzed to identify their natural GV biosynthetic elements. Based on the ultrasound screening method (VINNO D860LAB, 2D transducer X6-16L, 50 % output power mode, MI: 1.116), 57 seed resources with biosynthetic UMI probe potential from 10 genera were selected from 158 strains of cyanobacteria, corresponding to 28 types of effective biosynthetic elements. These results provide a theoretical basis for future mass production of customized GVs in chassis cells and their application in UMI.

Synthetic Element Chemistry based on Precise Designs of Reagents and Transition States

Synthetic Element Chemistry based on Precise Designs of Reagents and Transition States

Design and deep learning of synthetic B-cell-specific promoters.

Synthetic biology and deep learning synergistically revolutionize our ability for decoding and recoding DNA regulatory grammar. The B-cell-specific transcriptional regulation is intricate, and unlock the potential of B-cell-specific promoters as synthetic elements is important for B-cell engineering. Here, we designed and pooled synthesized 23640 B-cell-specific promoters that exhibit larger sequence space, B-cell-specific expression, and enable diverse transcriptional patterns in B-cells. By MPRA (Massively parallel reporter assays), we deciphered the sequence features that regulate promoter transcriptional, including motifs and motif syntax (their combination and distance). Finally, we built and trained a deep learning model capable of predicting the transcriptional strength of the immunoglobulin V gene promoter directly from sequence. Prediction of thousands of promoter variants identified in the global human population shows that polymorphisms in promoters influence the transcription of immunoglobulin V genes, which may contribute to individual differences in adaptive humoral immune responses. Our work helps to decipher the transcription mechanism in immunoglobulin genes and offers thousands of non-similar promoters for B-cell engineering.

Optimisation of TP53 reporters by systematic dissection of synthetic TP53 response elements.

TP53 is a transcription factor that controls multiple cellular processes, including cell cycle arrest, DNA repairand apoptosis. The relation between TP53 binding site architecture and transcriptional output is still not fully understood. Here, we systematically examined in three different cell lines the effects of binding site affinity and copy number on TP53-dependent transcriptional output, and also probed the impact of spacer length and sequence between adjacent binding sites, and of core promoter identity. Paradoxically, we found that high-affinity TP53 binding sites are less potent than medium-affinity sites. TP53 achieves supra-additive transcriptional activation through optimally spaced adjacent binding sites, suggesting a cooperative mechanism. Optimally spaced adjacent binding sites have a ∼10-bp periodicity, suggesting a role for spatial orientation along the DNA double helix. We leveraged these insights to construct a log-linear model that explains activity from sequence features, and to identify new highly active and sensitive TP53 reporters.

Temporally gene knockout using heat shock-inducible genome-editing system in plants.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) has emerged as a powerful tool to generate targeted loss-of-function mutations for functional genomic studies. As a next step, tools to generate genome modifications in a spatially and temporally precise manner will enable researchers to further dissect gene function. Here, we present two heat shock-inducible genome-editing (IGE) systems that efficiently edit target genes when the system is induced, thus allowing us to target specific developmental stages. For this conditional editing system, we chose the natural heat-inducible promoter from heat-shock protein 18.2 (HSP18.2) from Arabidopsis thaliana and the synthetic heat-inducible promoter heat shock-response element HSE-COR15A to drive the expression of Cas9. We tested these two IGE systems in Arabidopsis using cyclic or continuous heat-shock treatments at the seedling and bolting stages. A real-time quantitative polymerase chain reaction analysis revealed that the HSP18.2 IGE system exhibited higher Cas9 expression levels than the HSE-COR15A IGE system upon both cyclic and continuous treatments. By targeting brassinosteroid-insensitive 1 (BRI1) and phytoene desaturase (PDS), we demonstrate that both cyclic and continuous heat inductions successfully activated the HSP18.2 IGE system at the two developmental stages, resulting in highly efficient targeted mutagenesis and clear phenotypic outcomes. By contrast, the HSE-COR15A IGE system was only induced at the seedling stage and was less effective than the HSP18.2 IGE system in terms of mutagenesis frequencies. The presented heat shock-IGE systems can be conditionally induced to efficiently inactivate genes at any developmental stage and are uniquely suited for the dissection and systematic characterization of essential genes.

Production of Graft Copolymers of Cod Collagen with Butyl Acrylate and Vinyl Butyl Ether in the Presence of Triethylborane-Prospects for Use in Regenerative Medicine.

Collagen is a suitable material for regenerative medicine because it is characterized by its good biocompatibility. However, due to its fibrillar structure, it cannot organize itself into three-dimensional porous structures without additional modification. The introduction of synthetic monomer elements into the collagen macromolecules is a technique used to form three-dimensional, collagen-based, branched, and crosslinked structures. New types of graft copolymers made from cod collagen with a butyl acrylate and vinyl butyl ether copolymer in aqueous dispersion were obtained in the presence of triethylborane by a radical mechanism. The process of graft copolymer formation proceeded as usual by radical initiation, through radicals formed during triethylborane oxidation by oxygen residues, collagen borination, and reversible inhibition with the participation of a boroxyl radical. The characteristics of the graft copolymers were determined using methods of physical and chemical analysis (GPC, SEM, IR spectroscopy, etc.), while the cytotoxicity was assessed using the MTT assay method. It is shown that the grafting of alternating blocks of butyl acrylate and vinyl butyl ether to the protein macromolecules results in changes in the morphological pattern of the graft co-polymer in comparison with native collagen. This is manifested in the development of consolidations around the collagen fibers of the structural matrices, with the co-polymer cellular structure consisting of interpenetrating pores of unequal size. Additionally, it is important that the graft co-polymer solutions are not toxic at a certain concentration. The above properties confirm the promising nature of the technique's application as the basis for producing new materials for regenerative medicine.

Simulation of liquid production and water cut dynamics using fluid flow model and neural networks

In the oil industry, there is a noticeable tendency to use proxy modeling of various levels of complexity to perform operational predictive calculations, in particular machine learning methods that are actively developing in the context of digitalization and intellectualization of production processes. In this paper, using the example of a synthetic oil reservoir model development element, we present an approach to the joint use of a physically meaningful fluid flow model and machine learning methods for solving adaptation and prediction problems. A feature of the considered synthetic model is the presence of a pronounced zonal inhomogeneity of the permeability field. Within the framework of the proposed approach, a single-phase filtration model, simplified in comparison with the original formulation was used, the history matching of which was carried out by restoring the field of reservoir filtration parameters using a network of radial basis functions. Based on the reconstructed field, the connection coefficients between the wells were calculated, which qualitatively and quantitatively correspond to the true well connections. The next step was to train a recurrent neural network in order to predict the water cut of the produced fluid. The use of a recurrent neural network made it possible to reproduce the characteristic non-monotonic behavior of the water cut of the produced fluid, caused by non-stationary modes of operation of injection and production wells. A combination of the presented models makes it possible to predict the volume of the produced fluid and its phase composition. To assess the predictive properties of the models, the actual data set was divided into training and test intervals.

Stem-loop structures control mRNA processing of the cellulosomal cip-cel operon in Ruminiclostridium cellulolyticum

BackgroundAnaerobic, mesophilic, and cellulolytic Ruminiclostridium cellulolyticum produces an efficient cellulolytic extracellular complex named cellulosome, which consist of a non-catalytic multi-functional integrating subunit, organizing the various catalytic subunits into the complex. Main components of cellulosome were encoded by the cip-cel operon in R. cellulolyticum, and their stoichiometry is controlled by the mechanism of selective RNA processing and stabilization, which allows to confer each processed RNA portion from the cip-cel mRNA on different fates due to their stability and resolve the potential contradiction between the equimolar stoichiometry of transcripts with a within a transcription unit and the non-equimolar stoichiometry of subunits.ResultsIn this work, RNA processing events were found to occur at six intergenic regions (IRs) harboring stem-loop structures in cip-cel operon. These stem-loops not only stabilize processed transcripts at their both ends, but also act as cleavage signals specifically recognized by endoribonucleases. We further demonstrated that cleavage sites were often located downstream or 3′ end of their associated stem-loops that could be classified into two types, with distinct GC-rich stems being required for RNA cleavage. However, the cleavage site in IR4 was found to be located upstream of the stem-loop, as determined by the bottom AT-pair region of this stem-loop, together with its upstream structure. Thus, our findings reveal the structural requirements for processing of cip-cel transcripts, which can be potentially used to control the stoichiometry of gene expression in an operon.ConclusionsOur findings reveal that stem-loop structures acting as RNA cleavage signals not only can be recognized by endoribonucleases and determine the location of cleavage sites but also determine the stoichiometry of their flanking processed transcripts by controlling stability in cip-cel operon. These features represent a complexed regulation of cellulosome in the post-transcriptional level, which can be exploited for designing synthetic elements to control gene expression.

Development of Terminator-Promoter Bifunctional Elements for Application in Saccharomyces cerevisiae Pathway Engineering.

The construction of a genetic circuit requires the substitution and redesign of different promoters and terminators. The assembly efficiency of exogenous pathways will also decrease significantly when the number of regulatory elements and genes is increased. We speculated that a novel bifunctional element with promoter and terminator functions could be created via the fusion of a termination signal with a promoter sequence. In this study, the elements from a Saccharomyces cerevisiae promoter and terminator were employed to design a synthetic bifunctional element. The promoter strength of the synthetic element is apparently regulated through a spacer sequence and an upstream activating sequence (UAS) with a ~5-fold increase, and the terminator strength could be finely regulated by the efficiency element, with a ~5-fold increase. Furthermore, the use of a TATA box-like sequence resulted in the adequate execution of both functions of the TATA box and the efficiency element. By regulating the TATA box-like sequence, UAS, and spacer sequence, the strengths of the promoter-like and terminator-like bifunctional elements were optimally fine-tuned with ~8-fold and ~7-fold increases, respectively. The application of bifunctional elements in the lycopene biosynthetic pathway showed an improved pathway assembly efficiency and higher lycopene yield. The designed bifunctional elements effectively simplified pathway construction and can serve as a useful toolbox for yeast synthetic biology.

Simulated annealing framework for generating representative volume elements of materials with complex ligamentous microstructures

At the microscale, various materials from biological tissues to nanoporous metals are formed by networks of ligaments. Here we propose a highly efficient simulated annealing (SA) framework for generating synthetic representative volume elements (RVE) of such materials. It can produce RVE where the microstructural characteristics both on the network level (e.g., node valency and ligament length) and on the level of individual ligaments (e.g., curvature) can be predefined by the user via probability distributions. As an application example of our framework, we generate a large variety of RVEs, analyze their mechanical properties by the finite element method, and establish through this approach links between microstructural descriptors and macromechanical properties of materials with ligamentous microstructures.

Landmark and Tourist Spots Using Augmented Reality

It takes seriously a lot of time to build a system design like this, but why not use the rising technology to build an Augmented Reality design? Using the appropriate technology can help minimize the time needed in building one, plus it can show other people that society is catching up in terms of the new applications and technologies. For one, Augmented Reality (AR) is a technology that overlays digital information on a live view of the physical world to create a blended experience. Augmented Reality has now progressed to the point where real-time applications are considered and needed. Consequently, in this study, Augmented Reality is used to show the 3D model of the selected Landmark and the whole Municipality of Dumangas, Iloilo by just pointing the Android mobile device to the provided Landmark marker. However, it is important that synthetic elements are rendered and aligned in the scene in an accurate and visually acceptable way. In order to address these issues real-time, robust, and efficient model-based tracking was proposed for a mobile camera - a virtual map for the Municipality of Dumangas, Iloilo. This would help not only the Municipality of Dumangas to promote their tourist attraction but also the people who want to locate different tourist spots in Dumangas, Iloilo. This application would also provide the user with information about the specified Landmarks. The Landmark Tourist Spots Using Augmented Reality would test the user to know the Landmark in Dumangas, Iloilo.

Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum.

Cryptosporidium parvum is a high-risk and opportunistic waterborne parasitic pathogen with highly infectious oocysts that can survive harsh environmental conditions for long periods. Current state-of-the-art methods are limited to lengthy imaging and antibody-based detection techniques that are slow, labor-intensive, and demand trained personnel. Therefore, the development of new sensing platforms for rapid and accurate identification at the point-of-care (POC) is essential to improve public health. Herein, we propose a novel electrochemical microfluidic aptasensor based on hierarchical 3D gold nano-/microislands (NMIs), functionalized with aptamers specific to C. parvum. We used aptamers as robust synthetic biorecognition elements with a remarkable ability to bind and discriminate among molecules to develop a highly selective biosensor. Also, the 3D gold NMIs feature a large active surface area that provides high sensitivity and a low limit of detection (LOD), especially when they are combined with aptamers,. The performance of the NMI aptasensor was assessed by testing the biosensor's ability to detect different concentrations of C. parvum oocysts spiked in different sample matrices, i.e., buffer, tap water, and stool, within 40 min detection time. The electrochemical measurements showed an acceptable LOD of 5 oocysts mL-1 in buffer medium, as well as 10 oocysts mL-1 in stool and tap water media, over a wide linear range of 10-100,000 oocysts mL-1. Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibiting no significant cross-reactivity to other related coccidian parasites. The specific feasibility of the aptasensor was further demonstrated by the detection of the target C. parvum in patient stool samples. Our assay showed coherent results with microscopy and real-time quantitative polymerase chain reaction, achieving high sensitivity and specificity with a significant signal difference (p < 0.001). Therefore, the proposed microfluidic electrochemical biosensor platform could be a stepping stone for the development of rapid and accurate detection of parasites at the POC.

AGRICULTURAL INFLUENCE FACTORS ON THE ENVIRONMENTAL STATE OF THE LOW-PROTECTED FOREST STRIPS OF THE RIGHT-BANK FOREST STEPPE

Today, the current state of field protection forest plantations is practically unknown. This is due to the fact that their inventory has not been carried out since the mid-70s of the twentieth century. There are data indicating the presence of 0.43-0.44 million hectares of field protection plantations in Ukraine. About 13 million hectares of agricultural land are under the protection of this area of field protection forest strips, which is only 30% of the total area of such land in Ukraine. Taking into account roadside, farm, watershed forest strips, forest areas adjacent to fields, steppe glades with trees, the area of agrolandscape protection increases to 40% of their total number. Today, field protection forest cover in the agro-landscapes of Ukraine is 1.5%, while the scientifically-based coverage is 3.0-3.5%. Therefore, along with the creation of new forest strips, an important task is the inventory, care, reconstruction and preservation of the existing field protection forest plantations. Plowing the edges of the field to the outermost row of trees is most often 1.5 m for the main field protection forest strips and 1.0 m for the auxiliary field protection forest strips, although this distance can vary from 0.5 to 10.0 m. Among all the studied field protection forest strips 25,0-28.6% did not have field roads. Other field protection forest strips had field roads with an average width of 3.0-4.0 m. The projection of the crown of the outer row of trees of the forest strip protruding above the field can be 1.0-12.0 m, most often - 7.0-8.0 m. Corn and winter wheat are the main agricultural crops adjacent to field protection forest strips in the conditions of the Right Bank Forest Steppe. They were grown on 71.4-75.0% of the investigated fields. The main factors of suppressing the trees of field protection forest strips by cultivated crops are deep plowing to a depth of 25-27 cm near the forest strip for corn crops; the spreading method of applying mineral fertilizers, which is about 850 kg/ha of physical weight when growing corn and 650 kg/ha - when growing winter wheat; spraying of crops with synthetic pesticides, growth stimulants, trace elements and other drugs, which can be repeated up to 8-9 times during the growing season for both winter wheat and corn, especially by aerial method.

Computational modelling analysis and synthesis of molecularly imprinted polymers (MIPs) with two functional monomers using bulk polymerization

Sulfamethoxazole (SMX) is a well-known antibiotic used for the treatment and prevention of diseases for dairy-producing animals. However, the overuse of antibiotics in milk-producing herd resulted in detectable traces in milk which can cause severe effect to human health and also pose a risk of antimicrobial resistance through milk consumption. Therefore, a simple and rapid detection method is required to monitor the level of SMX in milk. Molecularly imprinted polymer (MIP) is a synthetic recognition element producing structurally artificial recognition sites “cavities” in polymeric matrices which are complementary to SMX structure for rebinding purposes through a process known as molecular imprinting. This method allows the capture of SMX with high affinity and selectivity even in the presence of antibiotics with very similar molecular structures to SMX. Herein, this study aims to synthesize and characterize MIP with specific binding to SMX through computational predictions and experimental verification using two different functional monomers which are methacrylic acid (MAA) and acrylamide (AA). The docking analysis using PyRx – Virtual Screening Tool showed higher binding affinity for SMX-MAA compared to SMX-AA which were −2.0 and −1.7 kcal/mol, respectively. Smaller binding affinity values indicate a greater interaction between the MIP and the analyte. This modelling data was further validated through experimental study by synthesizing MIP using MAA and AA monomers which presented an imprinting factor (IF) of 1.46 and 0.98 in ultra-pure water (UPW), and 1.02 and 0.93 in phosphate buffer solution (PBS) media respectively. IF values in both mediums showed higher affinity for SMX-MAA compared to SMX-AA and in agreement with the predicted values from the docking analysis. All in all, MIP synthesized using MAA as the monomer can be used for selective detection of SMX as a recognition element in sensors. This method is simple, stable and relatively cheap compared to the conventional detection methods such as immunoassays, GC/MS and HPLC.

Analysis of the Drosophila and human DPR elements reveals a distinct human variant whose specificity can be enhanced by machine learning.

The RNA polymerase II core promoter is the site of convergence of the signals that lead to the initiation of transcription. Here, we performed a comparative analysis of the downstream core promoter region (DPR) in Drosophila and humans by using machine learning. These studies revealed a distinct human-specific version of the DPR and led to the use of machine learning models for the identification of synthetic extreme DPR motifs with specificity for human transcription factors relative to Drosophila factors and vice versa. More generally, machine learning models could similarly be used to design synthetic DNA elements with customized functional properties.

Tunable control of insect pheromone biosynthesis in Nicotiana benthamiana.

Previous work has demonstrated that plants can be used as production platforms for molecules used in health, medicine, and agriculture. Production has been exemplified in both stable transgenic plants and using transient expression strategies. In particular, species of Nicotiana have been engineered to produce a range of useful molecules, including insect sex pheromones, which are valued for species-specific control of agricultural pests. To date, most studies have relied on strong constitutive expression of all pathway genes. However, work in microbes has demonstrated that yields can be improved by controlling and balancing gene expression. Synthetic regulatory elements that provide control over the timing and levels of gene expression are therefore useful for maximizing yields from heterologous biosynthetic pathways. In this study, we demonstrate the use of pathway engineering and synthetic genetic elements for controlling the timing and levels of production of Lepidopteran sex pheromones in Nicotiana benthamiana. We demonstrate that copper can be used as a low-cost molecule for tightly regulated inducible expression. Further, we show how construct architecture influences relative gene expression and, consequently, product yields in multigene constructs. We compare a number of synthetic orthogonal regulatory elements and demonstrate maximal yields from constructs in which expression is mediated by dCas9-based synthetic transcriptional activators. The approaches demonstrated here provide new insights into the heterologous reconstruction of metabolic pathways in plants.

Abstract 2918: In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice

Abstract Introduction: Direct in vivo delivery of lentiviral vectors (LV) to generate CD19 CAR+ cells without the need for ex vivo preparation represents a promising approach to transform autologous CAR therapy into an off-the-shelf treatment. In these studies, direct administration of a new LV encoding a CD19 CAR into humanized NOD SCID gamma (NSG) mice expressing human IL-3, GM-CSF, and SCF (NSG-SGM3) resulted in a dose-dependent elimination of B cells in the peripheral blood, peritoneal fluid, bone marrow, and tissue of treated mice. Methods: CD3-directed LV encoding a CD19 CAR with a novel synthetic driver element was manufactured utilizing a 25L clinical scale suspension-based process. NSG-SGM3 mice transplanted with human CD34+ cells from cord blood were injected with LV doses (1E6 IU, 1E7 IU, or 5E7 IU) intraperitoneally (IP) or 1E7 IU intravenously (IV). Quantification of CD19 CAR+ cells and CD20+ B cells in peripheral blood, peritoneal fluid, and bone marrow was assessed by flow cytometry. Additionally, immunohistochemical analysis was performed to evaluate the tissue-resident human B cells and for any other histopathological observations following test article administration. Results: All CD34+ humanized NSG-SGM3 mice were confirmed to exhibit efficient human hematopoietic engraftment by flow cytometry prior to test article administration (peripheral blood hCD45: 68.9% ± 4.93%; hCD19+ B cells: 53.7% ± 5.11%). Direct LV administration at the 1E7 IU and 5E7 IU doses demonstrated a dose-dependent reduction in circulating human B cells compared to the control and 1E6 IU dose (p &amp;lt; 0.05). The synthetic driver elements co-expressed with the CAR led to the formation of unique CD3+ CD8+ CD56+ T and NK-like (TaNK) CD19 CAR+ cells in circulation. The 1E7 IU IP dose demonstrated ablation of B cells (total cells/uL) in peripheral blood (control: 9.67 ± 3.72 vs. LV treated: 0.157 ± 0.117), intraperitoneal fluid (control: 0.322 ± 0.244 vs. LV treated: 0.038 ± 0.029), bone marrow (control: 9.16± 1.83 vs. LV treated: 0.734 ± 0.864), and splenic tissue. Both IP and IV routes of administration showed significant B cell depletion at the 1E7 IU dose. However, complete B cell elimination in splenic tissue was only observed at the 5E7 IU dose. Non-treated CD34+ humanized NSG-SGM3 mice exhibited hepatic portal inflammation and moderate graft-versus-host disease (GVHD) in the colon. Interestingly, mice treated with LV encoding CD19 CAR exhibited decreased inflammatory pathology, suggesting potential B cell involvement in the inflammatory response in this model. Conclusion: In this study, direct in vivo delivery of LV encoding CD19 CAR resulted in the generation of functionally active CD19 CAR TaNK cells capable of eliminating target B cells in peripheral blood, peritoneal fluid, bone marrow, and tissue. Citation Format: Frederic Vigant, Ani Kundu, Ramya Yarlagadda, Cody Gowan, Michael Betts, Jonathan Kato, Renata Soares, Alan Ponce, Lintao Liu, Junyi Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Suraj Kachgal, Gregory Schreiber, Wei Zhang, Gregory Wade, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2918.

A self‐propagating, barcoded transposon system for the dynamic rewiring of genomic networks

In bacteria, natural transposon mobilization can drive adaptive genomic rearrangements. Here, we build on this capability and develop an inducible, self‐propagating transposon platform for continuous genome‐wide mutagenesis and the dynamic rewiring of gene networks in bacteria. We first use the platform to study the impact of transposon functionalization on the evolution of parallel Escherichia coli populations toward diverse carbon source utilization and antibiotic resistance phenotypes. We then develop a modular, combinatorial assembly pipeline for the functionalization of transposons with synthetic or endogenous gene regulatory elements (e.g., inducible promoters) as well as DNA barcodes. We compare parallel evolutions across alternating carbon sources and demonstrate the emergence of inducible, multigenic phenotypes and the ease with which barcoded transposons can be tracked longitudinally to identify the causative rewiring of gene networks. This work establishes a synthetic transposon platform that can be used to optimize strains for industrial and therapeutic applications, for example, by rewiring gene networks to improve growth on diverse feedstocks, as well as help address fundamental questions about the dynamic processes that have sculpted extant gene networks.

Shoot-root signal circuit: Phytoremediation of heavy metal contaminated soil.

High concentrations of heavy metals in the environment will cause serious harm to ecosystems and human health. It is urgent to develop effective methods to control soil heavy metal pollution. Phytoremediation has advantages and potential for soil heavy metal pollution control. However, the current hyperaccumulators have the disadvantages of poor environmental adaptability, single enrichment species and small biomass. Based on the concept of modularity, synthetic biology makes it possible to design a wide range of organisms. In this paper, a comprehensive strategy of "microbial biosensor detection - phytoremediation - heavy metal recovery" for soil heavy metal pollution control was proposed, and the required steps were modified by using synthetic biology methods. This paper summarizes the new experimental methods that promote the discovery of synthetic biological elements and the construction of circuits, and combs the methods of producing transgenic plants to facilitate the transformation of constructed synthetic biological vectors. Finally, the problems that should be paid more attention to in the remediation of soil heavy metal pollution based on synthetic biology were discussed.

Mobility of mPing and its associated elements is regulated by both internal and terminal sequences

BackgroundDNA transposable elements are mobilized by a “cut and paste” mechanism catalyzed by the binding of one or more transposase proteins to terminal inverted repeats (TIRs) to form a transpositional complex. Study of the rice genome indicates that the mPing element has experienced a recent burst in transposition compared to the closely related Ping and Pong elements. A previously developed yeast transposition assay allowed us to probe the role of both internal and terminal sequences in the mobilization of these elements.ResultsWe observed that mPing and a synthetic mPong element have significantly higher transposition efficiency than the related autonomous Ping and Pong elements. Systematic mutation of the internal sequences of both mPing and mPong identified multiple regions that promote or inhibit transposition. Simultaneous alteration of single bases on both mPing TIRs resulted in a significant reduction in transposition frequency, indicating that each base plays a role in efficient transposase binding. Testing chimeric mPing and mPong elements verified the important role of both the TIRs and internal regulatory regions. Previous experiments showed that the G at position 16, adjacent to the 5′ TIR, allows mPing to have higher mobility. Alteration of the 16th and 17th base from mPing’s 3′ end or replacement of the 3′ end with Pong 3′ sequences significantly increased transposition frequency.ConclusionsAs the transposase proteins were consistent throughout this study, we conclude that the observed transposition differences are due to the element sequences. The presence of sub-optimal internal regions and TIR bases supports a model in which transposable elements self-limit their activity to prevent host damage and detection by host regulatory mechanisms. Knowing the role of the TIRs, adjacent sub-TIRs, and internal regulatory sequences allows for the creation of hyperactive elements.