CRISPRi-mediated tunable control of gene expression level with engineered single-guide RNA in Escherichia coli.

Precise control of gene expression is essential for flux redistribution in metabolic pathways. Although the CRISPR interference (CRISPRi) system can effectively repress gene expression at the transcriptional level, it has still been difficult to precisely control the level without loss of specificity or an increase in cell toxicity. In this study, we developed a tunable CRISPRi system that performs transcriptional regulation at various levels. We constructed a single-guide RNA (sgRNA) library targeting repeat, tetraloop, and anti-repeat regions to modulate the binding affinity against dCas9. Each screened sgRNA could regulate the gene expression at a certain level between fully-repressing and non-repressing states (>45-fold). These sgRNAs also enabled modular regulation with various target DNA sequences. We applied this system to redistribute the metabolic flux to produce violacein derivatives in a predictable ratio and optimize lycopene production. This system would help accelerate the flux optimization processes in metabolic engineering and synthetic biology.

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

Synthetic protein quality control to enhance full-length translation in bacteria.

Coupled transcription and translation processes in bacteria cause indiscriminate translation of intact and truncated messenger RNAs, inevitably generating nonfunctional polypeptides. Here, we devised a synthetic protein quality control (ProQC) system that enables translation only when both ends of mRNAs are present and followed by circularization based on sequence-specific RNA-RNA hybridization. We demonstrate that the ProQC system dramatically improved the fraction of full-length proteins among all synthesized polypeptides by selectively translating intact mRNA and reducing abortive translation. As a result, full-length protein synthesis increased up to 2.5-fold without changing the transcription or translation efficiency. Furthermore, we applied the ProQC system for 3-hydroxypropionic acid, violacein and lycopene production by ensuring full-length expression of enzymes in biosynthetic pathways, resulting in 1.6- to 2.3-fold greater biochemical production. We believe that our ProQC system can be universally applied to improve not only the quality of recombinant protein production but also efficiencies of metabolic pathways.

Hydrogel Micropillar Array for Temperature Sensing in Fluid.

Localized temperature sensing and control on a micron-scale have diverse applications in biological systems. We present a micron-sized hydrogel pillar array as potential temperature probes and actuators by exploiting sensitive temperature dependence of their volume change. Soft lithography-based molding processes were presented to fabricate poly N-isopropyl acrylamide (p-NIPAAm)-based hydrogel pillar array on a glass substrate. Au nanorods as light-induced heating elements were embedded within the hydrogel pillars, and near-infrared (NIR) light was used to modulate temperature in a local area. First, static responses of the micro-pillar array were characterized as a function of its temperature. It was shown that the hydrogel had a sensitive volume transition near its low critical solution temperature (LCST). Furthermore, we showed that LCST could be readily adjusted by utilizing copolymerizing with acrylamide (AAM). To demonstrate the feasibility of spatiotemporal temperature mapping and modulation using the presented pillar array, pulsed NIR light was illuminated on a local area of the hydrogel pillar array, and its responses were recorded. Dynamic temperature change in water was mapped based on the abrupt volume change characteristics of the hydrogel pillar, and its potential actuation using NIR light was successfully demonstrated. Considering that the structure can be arrayed in a two-dimensional pixel format with high spatial resolution and high sensitive temperature characteristics, the presented method and the device structure can have diverse applications to change and sense local temperatures in liquid. This is particularly useful in biological systems, where their physiological temperature can be modulated and mapped with high spatial resolution.

Cellular responses to reactive oxygen species are predicted from molecular mechanisms.

Catalysis using iron-sulfur clusters and transition metals can be traced back to the last universal common ancestor. The damage to metalloproteins caused by reactive oxygen species (ROS) can prevent cell growth and survival when unmanaged, thus eliciting an essential stress response that is universal and fundamental in biology. Here we develop a computable multiscale description of the ROS stress response in Escherichia coli, called OxidizeME. We use OxidizeME to explain four key responses to oxidative stress: 1) ROS-induced auxotrophy for branched-chain, aromatic, and sulfurous amino acids; 2) nutrient-dependent sensitivity of growth rate to ROS; 3) ROS-specific differential gene expression separate from global growth-associated differential expression; and 4) coordinated expression of iron-sulfur cluster (ISC) and sulfur assimilation (SUF) systems for iron-sulfur cluster biosynthesis. These results show that we can now develop fundamental and quantitative genotype-phenotype relationships for stress responses on a genome-wide basis.

Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.

Two major transcriptional regulators of carbon metabolism in bacteria are Cra and CRP. CRP is considered to be the main mediator of catabolite repression. Unlike for CRP, in vivo DNA binding information of Cra is scarce. Here we generate and integrate ChIP-exo and RNA-seq data to identify 39 binding sites for Cra and 97 regulon genes that are regulated by Cra in Escherichia coli. An integrated metabolic-regulatory network was formed by including experimentally-derived regulatory information and a genome-scale metabolic network reconstruction. Applying analysis methods of systems biology to this integrated network showed that Cra enables optimal bacterial growth on poor carbon sources by redirecting and repressing glycolysis flux, by activating the glyoxylate shunt pathway, and by activating the respiratory pathway. In these regulatory mechanisms, the overriding regulatory activity of Cra over CRP is fundamental. Thus, elucidation of interacting transcriptional regulation of core carbon metabolism in bacteria by two key transcription factors was possible by combining genome-wide experimental measurement and simulation with a genome-scale metabolic model.

Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655.

Transcriptional regulation enables cells to respond to environmental changes. Of the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, 185 have been experimentally identified, but ChIP methods have been used to fully characterize only a few dozen. Identifying these remaining TFs is key to improving our knowledge of the E. coli transcriptional regulatory network (TRN). Here, we developed an integrated workflow for the computational prediction and comprehensive experimental validation of TFs using a suite of genome-wide experiments. We applied this workflow to (i) identify 16 candidate TFs from over a hundred uncharacterized genes; (ii) capture a total of 255 DNA binding peaks for ten candidate TFs resulting in six high-confidence binding motifs; (iii) reconstruct the regulons of these ten TFs by determining gene expression changes upon deletion of each TF and (iv) identify the regulatory roles of three TFs (YiaJ, YdcI, and YeiE) as regulators of l-ascorbate utilization, proton transfer and acetate metabolism, and iron homeostasis under iron-limited conditions, respectively. Together, these results demonstrate how this workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs in parallel.

Rapid photothermal actuation of light-addressable, arrayed hydrogel columns in a macroporous silicon membrane.

This paper presents rapid photothermal actuation of light-addressable, arrayed hydrogel columns in a macroporous silicon membrane. Au nanorods are incorporated into thermo-responsive p-NIPAAm hydrogel to utilize surface plasmon-induced local heating by near-infrared light. By measuring optical transmission through the fabricated membrane structure with Au nanorod embedded hydrogel, we have demonstrated that photothermal actuation of hydrogel can be done in two-dimensional, pixel-like configuration with high spatial and temporal resolutions. Benefiting from the hydrogel volume confinement within micron-sized pores, we have achieved sub-second response time of hydrogel photothermal actuation and its repeatable photothermal actuation on highly localized illuminated area. Considering that each hydrogel column is confined within each pore and different wavelength of light can be used to induce photothermal actuation of hydrogel's deswelling characteristics by modifying physical dimensions of Au nanorods, it has a potential for optically-addressable, multiplexed drug release systems with rapid response time.

Transcriptional Profiling of the Probiotic Escherichia coli Nissle 1917 Strain under Simulated Microgravity.

Long-term space missions affect the gut microbiome of astronauts, especially the viability of some pathogens. Probiotics may be an effective solution for the management of gut microbiomes, but there is a lack of studies regarding the physiology of probiotics in microgravity. Here, we investigated the effects of microgravity on the probiotic Escherichia coli Nissle 1917 (EcN) by comparing transcriptomic data during exponential and stationary growth phases under simulated microgravity and normal gravity. Microgravity conditions affected several physiological features of EcN, including its growth profile, biofilm formation, stress responses, metal ion transport/utilization, and response to carbon starvation. We found that some changes, such as decreased adhesion ability and acid resistance, may be disadvantageous to EcN relative to gut pathogens under microgravity, indicating the need to develop probiotics optimized for space flight.

dCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene for Liquid Biopsy.

The-state-of-the-art bio- and nanotechnology have opened up an avenue to noninvasive liquid biopsy for identifying diseases from biomolecules in bloodstream, especially DNA. In this work, we combined sequence-specific-labeling scheme using mutated clustered regularly interspaced short palindromic repeats associated protein 9 without endonuclease activity (CRISPR/dCas9) and ion concentration polarization (ICP) phenomenon as a mechanism to selectively preconcentrate targeted DNA molecules for rapid and direct detection. Theoretical analysis on ICP phenomenon figured out a critical mobility, elucidating two distinguishable concentrating behaviors near a nanojunction, a stacking and a propagating behavior. Through the modulation of the critical mobility to shift those behaviors, the C-C chemokine receptor type 5 ( CCR5) sequences were optically detected without PCR amplification. Conclusively, the proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and accurate micro/nanofluidic platform of liquid biopsies for disease diagnostics.

Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

Optical and Acoustic Sensor-Based 3D Ball Motion Estimation for Ball Sport Simulators †.

Estimation of the motion of ball-shaped objects is essential for the operation of ball sport simulators. In this paper, we propose an estimation system for 3D ball motion, including speed and angle of projection, by using acoustic vector and infrared (IR) scanning sensors. Our system is comprised of three steps to estimate a ball motion: sound-based ball firing detection, sound source localization, and IR scanning for motion analysis. First, an impulsive sound classification based on the mel-frequency cepstrum and feed-forward neural network is introduced to detect the ball launch sound. An impulsive sound source localization using a 2D microelectromechanical system (MEMS) microphones and delay-and-sum beamforming is presented to estimate the firing position. The time and position of a ball in 3D space is determined from a high-speed infrared scanning method. Our experimental results demonstrate that the estimation of ball motion based on sound allows a wider activity area than similar camera-based methods. Thus, it can be practically applied to various simulations in sports such as soccer and baseball.

Comparative study of macroporous silicon-based photovoltaic characteristics using indium tin oxide-silicon and pn-silicon junction based devices.

We report highly ordered macroporous silicon (Si)-based photovoltaic characteristics using indium tin oxide (ITO)/n-Si and pn-Si junction-based devices. The detailed fabrication processes including new controlled ITO etching are presented. Theoretical device simulations are performed to understand the presented device structures and propose an optimum device design based on processing limitations. The performance of ITO/n-Si junction devices directly depends on the conformal ITO coating along the pore surface. While pn-Si junction device requires additional doping step, the device can overcome the limitation of ITO conformal coating, especially for a device with high-aspect-ratio macropore structures. Experimental results also support the simulation analysis. The three-dimensional structural properties of well-defined macroporous Si coupled with the formation of photovoltaic devices are attractive for multi-functional applications.

Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli.

Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression along with the deciphering of regulation of the related enzymes and an analogue transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli expressing the ALA biosynthetic pathway. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5'-untranslated regions (5'-UTRs). Then, the tricarboxylic acid (TCA) cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. Although the resulting strain showed a severe metabolic imbalance and low ALA production, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production. Thus, this precise tuning of the glyoxylate cycle in a quantitative manner should also enable efficient production of other value-added products derived from the TCA cycle.

Highly-stable and -flexible graphene/(CF3SO2)2NH/graphene transparent conductive electrodes for organic solar cells.

We first employ highly-stable and -flexible (CF3SO2)2NH-doped graphene (TFSA/GR) and GR-encapsulated TFSA/GR (GR/TFSA/GR) transparent conductive electrodes (TCEs) prepared on polyethylene terephthalate substrates for flexible organic solar cells (OSCs). Compared to conventional indium tin oxide (ITO) TCEs, the TFSA-doped-GR TCEs show higher optical transmittance and larger sheet resistance. The TFSA/GR and GR/TFSA/GR TCEs show work functions of 4.89 ± 0.16 and 4.97 ± 0.18 eV, respectively, which are not only larger than those of the ITO TCEs but also indicate p-type doping of GR, and are therefore more suitable for anode TCEs of OSCs. In addition, typical GR/TFSA/GR-TCE OSCs are much more mechanically flexible than the ITO-TCE ones with their photovoltaic parameters being similar, as proved by bending tests as functions of cycle and curvature.

An acousto-optic sensor based on resonance grating waveguide structure.

This paper presents an acousto-optic (AO) sensor based on resonance grating waveguide structure. The sensor is fabricated using elastic polymer materials to achieve a good sensitivity to ultrasound pressure waves. Ultrasound pressure waves modify the structural parameters of the sensor and result in the optical resonance shift of the sensor. This converts into a light intensity modulation. A commercial ultrasound transducer at 20 MHz is used to characterize a fabricated sensor and detection sensitivity at different optical source wavelength within a resonance spectrum is investigated. Practical use of the sensor at a fixed optical source wavelength is presented. Ultimately, the geometry of the planar sensor structure is suitable for two-dimensional, optical pressure imaging applications such as pressure wave detection and mapping, and ultrasound imaging.

Natural transformation of Vibrio natriegens with large genetic cluster enables alginate assimilation for isopentenol production.

Alginate is a major component of brown macroalgae, and its efficient utilization is critical for developing sustainable technologies. Vibrio natriegens is a fast-growing marine bacterium that has gained massive attention due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, V. natriegens was engineered to utilize marine biomass, kelp, as a carbon source. A total of 33.8 kb of the genetic cluster for alginate assimilation from Vibrio sp. dhg was integrated into V. natriegens by natural transformation. Engineered V. natriegens was further modified to produce 1.8 mg/L of isopentenol from 16 g/L of crude kelp powder. This study not only presents the very first case in which V. natriegens can be naturally transformed with large DNA fragments but also highlights the potential of this strain for converting marine biomass into valuable products.

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria.

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.

Model-driven rebalancing of the intracellular redox state for optimization of a heterologous n-butanol pathway in Escherichia coli.

The intracellular redox state plays an important role in the cellular physiology that determines the efficiency of chemical and biofuel production by microbial cell factories. However, it is difficult to achieve optimal redox rebalancing of synthetic pathways owing to the sensitive responses of cellular physiology according as the intracellular redox state changes. Here, we demonstrate optimal rebalancing of the intracellular redox state by model-driven control of expression using n-butanol production in Escherichia coli as a model system. The synthetic n-butanol production pathway was constructed by implementing synthetic constitutive promoters and designing synthetic 5'-untranslated regions (5'-UTR) for each gene. Redox rebalancing was achieved by anaerobically activating the pyruvate dehydrogenase (PDH) complex and additionally tuning the expression level of NAD(+)-dependent formate dehydrogenase (fdh1 from Saccharomyces cerevisiae) through rational UTR engineering. Interestingly, efficient production of n-butanol required different amounts of reducing equivalents depending on whether the substrate was glucose or galactose. One intriguing implication of this work is that additional strain improvement can be achieved, even within given genetic components, through rebalancing intracellular redox state according to target products and substrates.

Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency.

Precise prediction of prokaryotic translation efficiency can provide valuable information for optimizing bacterial host for the production of biochemical compounds or recombinant proteins. However, dynamic changes in mRNA folding throughout translation make it difficult to assess translation efficiency. Here, we systematically determined the universal folding regions that significantly affect the efficiency of translation in Escherichia coli. By assessing the specific regions for mRNA folding, we could construct a predictive design method, UTR Designer, and demonstrate that proper codon optimization around the 5'-proximal coding sequence is necessary to achieve a broad range of expression levels. Finally, we applied our method to control the threshold value of input signals switching on a genetic circuit. This should increase our understanding of the processes underlying gene expression and provide an efficient design principle for optimizing various biological systems, thereby facilitating future efforts in metabolic engineering and synthetic biology.