Development of a bacterial gene transcription activating strategy based on transcriptional activator positive feedback.

INTRODUCTION: Transcription of biological nitrogen fixation (nif) genes is activated by the NifA protein which recognizes specific activating sequences upstream of σ54-dependent nif promoters. The large quantities of nitrogenase which can make up 20% of the total proteins in the cell indicates high transcription activating efficiency of NifA and high transcription level of nifHDK nitrogenase genes. OBJECTIVES: Development of an efficient gene transcription activating strategy in bacteria based on positive transcription regulatory proteins and their regulating DNA sequences. METHODS: We designed a highly efficient gene transcription activating strategy in which the nifA gene was placed directly downstream of its regulating sequences. The NifA protein binds its regulating sequences and stimulates transcription of itself and downstream genes. Overexpressed NifA causes transcription activation by positive reinforcement. RESULTS: When this gene transcription activating strategy was used to overexpress NifA in Pseudomonas stutzeri DSM4166 containing the nif gene cluster, the nitrogenase activity was increased by 368 folds which was 16 times higher than that obtained by nifA driven by the strongest endogenous constitutive promoter. When this strategy was used to activate transcription of exogenous biosynthetic genes for the plant auxin indole-3-acetic acid and the antitumor alkaloid pigment prodigiosin in DSM4166, both of them resulted in better performance than the strongest endogenous constitutive promoter and the highest reported productions in heterologous hosts to date. Finally, we demonstrated the universality of this strategy using the positive transcriptional regulator of the psp operon, PspF, in E. coli and the pathway-specific positive transcription regulator of the polyene antibiotic salinomycin biosynthesis, SlnR, in Streptomyces albus. CONCLUSION: Many positive transcription regulatory proteins and their regulating DNA sequences have been identified in bacteria. The gene transcription activating strategy developed in this study will have broad applications in molecular biology and biotechnology.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity.

BACKGROUND: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. RESULTS: Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. CONCLUSIONS: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Polymerase/Nicking Enzyme Powered Dual-template Multi-cycle G-Triplex Machine for HIV-1 Determination.

We proposed a dual-template, multi-cycle DNA nanomachine driven by polymerase and a nicking enzyme with high efficiency. The reaction system simply consists of two templates (T1, T2) and two enzymes (KF polymerase, Nb.BbvCI). The two templates are similar in structure (X-X'-Y, Y-Y'-C) with a primer recognition region, a primer analogue generation region, an output region (3'-5'), and two nicking sites. The output strand of T1 is the primer of T2, and the G-rich fragment (G3) is designed as the final product. In the presence of HIV-1, numerous G3 were generated through the multi-cycle amplification strategy and formed a G-triplex/ThT complex after the addition of thioflavin T (ThT), which greatly enhanced the fluorescence intensity as a signal reporter in the label-free sensing strategy. A dynamic response range of 50 fM - 2 nM for HIV-1 gene detection can be achieved through this multi-cycle G-triplex machine, and benefiting from the high efficiency amplification strategy, the enzymatic reaction can be completed within 45 min and followed by fluorescence measurements. In addition, the analysis of other targets can be achieved by replacing the template sequence. Thus, there is a certain application potential of this strategy for trace biomarker analysis.

Multifunctional electrochemical biosensor with "tetrahedral tripods" assisted multiple tandem hairpins assembly for ultra-sensitive detection of target DNA.

Nucleic acids are genetic materials in the human body that play important roles in storing, copying, and transmitting genetic information. Abnormal nucleic acid sequences, base mutations, and genetic changes often lead to cancer and other diseases. Meanwhile, methylated DNA is one of the main epigenetic modifications, which is considered to be an excellent biomarker in the early detection, prognosis, and treatment of cancers. Therefore, a multifunctional electrochemical biosensor was constructed with sturdy tetrahedral tripods, which assisted multiple tandem hairpins through base complementary pairing and effective ultra-sensitive detection of targets (DNA, microRNA, and methylated DNA). In the experiments, experimental conditions were optimized, and different DNA concentrations in serum were detected to verify the sensitivity of the biosensor and the feasibility of this protocol. In addition, microRNA and DNA methylation were detected through different designs of tetrahedral tripods (TTs) that capture probes to prove the superiority of this scheme. A sturdy pyramid structure of TTs extremely enhanced the capture efficiency of targets. The targets triggered the one-step isothermal multi-tandem amplification reaction by incubating multiple hairpin assemblies. To our knowledge, a combination of two parts, which greatly reduced background interference and decreased non-specific substance interference, has appeared for the first time in this paper. Moreover, the load area of electrochemical substances was significantly increased than that in previous studies. This greatly increased the detection range and detection limit of targets. The electrochemical signal responses were generated in freely diffusing hexaammineruthenium(iii) chloride (RuHex). RuHex could adhere to the DNA phosphate backbone by a powerful electrostatic attraction, causing increased current responses.

Two-layer three-dimensional DNA walker with highly integrated entropy-driven and enzyme-powered reactions for HIV detection.

Here, we propose a new two-layer three-dimensional (3-D) DNA walker sensor with highly integrated entropy-driven and enzyme-powered reactions for the first time. The 3-D DNA walker sensor is constructed by assembling densely carboxyfluorescein-labeled single strand oligonucleotides (inner-layer tracks) and nucleic acid complex S (outer-layer tracks) on a microparticle. In the presence of the target, outer and inner tracks are activated in turn, thereby releasing a great deal of the signal reporters for signal reading. As a result, our 3-D DNA walker sensor can realize the target detection in the range from 2 pM to 5 nM within one hour. Besides, the specific walker sensor can clearly distinguish even one-base mismatched target analogue. More importantly, our walker sensor can also test the target in human serum samples in the concentrations as low as 0.1 nM, which provides a bridge between real sample detection and clinical application. Certainly, this smart strategy could also be generalized to any target of interest by proper design.

Symmetric exponential amplification reaction-based DNA nanomachine for the fluorescent detection of nucleic acids.

By introducing palindromic sequences into the classical exponential amplification reaction (EXPAR), we constructed a new palindromic fragment-incorporated multifunctional hairpin probe (P-HP)-mediated symmetric exponential amplification reaction (S-EXPAR), to significantly reduce the background signal caused by inherent nonspecific amplification. A G-triplex/ThT complex was used as the signal reporter for the proposed label-free DNA nanomachine. The P-HP consists of five functional regions: a C-rich region (C), a target DNA recognition region (T'), two nicking sites (X') and a palindromic fragment (P). When target DNA (T) hybridizes with P-HP, the palindromic fragment at the 3' end of P-HP is fully exposed. Then, the P-HP/T duplexes hybridize with each other through the exposed P, and EXPAR occurs automatically and continuously on both sides of P under the synergistic effect of polymerase and nicking endonuclease. This is called the S-EXPAR assay. In this system, one T converts to a large number of G-triplex fragments, which can combine with ThT within a short time. The G-triplex/ThT complexes formed act as the signal reporter in a label-free and environmentally friendly format. In this way, the limit of detection of this method is as low as 10 pM with a dynamic response range of 10 pM to 300 nM. In addition, this method can detect other nucleic acids by simply changing the T' region of the P-HP. Thus, the proposed DNA nanomachine is a potential alternative method for nucleic acid detection.

Enhanced Heterologous Spinosad Production from a 79-kb Synthetic Multioperon Assembly.

Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.

An electrochemical biosensor for microRNA-196a detection based on cyclic enzymatic signal amplification and template-free DNA extension reaction with the adsorption of methylene blue.

A simple and sensitive electrochemical biosensor was developed for microRNA-196a detection, which is of important diagnostic significance for pancreatic cancer. It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction. In the presence of microRNA-196a, duplex-specific nuclease (DSN) catalyzed the digestion of the 3'-PO4 terminated capture probe (CP), resulting in the target recycling amplification. Meanwhile, the 3'-OH terminal of CP was exposed. Then, template-free DNA extension reaction was triggered by terminal deoxynucleotidyl transferase (TdT), producing amounts of single-stranded DNA (ssDNA). After ssDNA absorbed numerous methylene blue (MB), an ultrasensitive electrochemical readout was obtained. Based on this dual amplification mechanism, the proposed biosensor exhibited a high sensitivity for detection of microRNA-196a down to 15 aM with a linear range from 0.05 fM to 50 pM. This biosensor displayed high specificity, which could discriminate target microRNAs from one base mismatched microRNAs. It also showed good reproducibility and stability. Furthermore, it was successfully applied to the determination of microRNA-196a in plasma samples. In conclusion, with the excellent analytical performance, this biosensor might have the potential for application in clinical diagnostics of pancreatic cancer.