Electrophysiological insights into the relationship between calcium dynamics and cardiomyocyte beating function in chronic hemodialysis treatment.

For patients in which the Ca2+ concentration of dialysis fluid is lower than that in plasma, chronic hemodialysis treatment often leads to cardiac beating dysfunction. By applying these conditions to an electrophysiological mathematical model, we evaluated the impact of body fluid Ca2+ dynamics during treatment on cardiomyocyte beating and, moreover, explored measures that may prevent cardiomyocyte beating dysfunction. First, Ca2+ concentrations in both plasma and interstitial fluid were decreased with treatment time, which induced both a slight decline in beating rhythm on a sinoatrial nodal cell and a wane in contraction force on a ventricular cell. These simulated results were in agreement with clinical observations. Next, a relationship between the intracellular Ca2+ concentration and ion current dynamics of ion transporters were examined to elucidate the mechanism underlying cardiomyocyte beating dysfunction. The inward current of the Na/Ca exchanger (NCX) increased with a decrease in Ca2+ concentration in interstitial fluid and induced a reduction in intracellular Ca2+ concentration during treatment. Furthermore, the decline in intracellular Ca2+ concentration reduced the contraction force. These findings implied that ion transport through the NCX is a dominant factor that induces cardiomyocyte beating dysfunction during hemodialysis. Finally, the replenishment of Ca2+ or application of an NCX inhibitor during treatment suppressed the decrease in intracellular Ca2+ concentration and contributed to the stabilization of cardiomyocyte beating function. In summary, the clinical implementation of hepatically cleared NCX inhibitor may be a suitable approach to improving the quality of life for patients on chronic hemodialysis.

Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942.

Genetic manipulation in cyanobacteria enables the direct production of valuable chemicals from carbon dioxide. However, there are still very few reports of the production of highly effective photosynthetic chemicals. Several synthetic metabolic pathways (e.g., isopropanol, acetone, isoprene, and fatty acids) have been constructed by branching from acetyl-CoA and malonyl-CoA, which are key intermediates for photosynthetic chemical production downstream of pyruvate decarboxylation. Recent reports of the absolute determination of cellular metabolites in Synechococcus elongatus PCC 7942 have shown that its acetyl-CoA levels corresponded to about one hundredth of the pyruvate levels. In short, one of the reasons for lower photosynthetic chemical production from acetyl-CoA and malonyl-CoA was the smaller flux to acetyl-CoA. Pyruvate decarboxylation is a primary pathway for acetyl-CoA synthesis from pyruvate and is mainly catalyzed by the pyruvate dehydrogenase complex (PDHc). In this study, we tried to enhance the flux toward acetyl-CoA from pyruvate by overexpressing PDH genes and, thus, catalyzing the conversion of pyruvate to acetyl-CoA via NADH generation. The overexpression of PDH genes cloned from S. elongatus PCC 7942 significantly increased PDHc enzymatic activity and intracellular acetyl-CoA levels in the crude cell extract. Although growth defects were observed in overexpressing strains of PDH genes, the combinational overexpression of PDH genes with the synthetic metabolic pathway for acetate or isopropanol resulted in about 7-fold to 9-fold improvement in its production titer, respectively (9.9 mM, 594.5 mg/L acetate, 4.9 mM, 294.5 mg/L isopropanol). PDH genes overexpression would, therefore, be useful not only for the production of these model chemicals, but also for the production of other chemicals that require acetyl-CoA as a key precursor.

Synthetic microbial consortium with specific roles designated by genetic circuits for cooperative chemical production.

Synthetic microbial consortia consisting of microorganisms with different synthetic genetic circuits or divided synthetic metabolic pathway components can exert functions that are beyond the capacities of single microorganisms. However, few consortia of microorganisms with different synthetic genetic circuits have been developed. We designed and constructed a synthetic microbial consortium composed of an enzyme-producing strain and a target chemical-producing strain using Escherichia coli for chemical production with efficient saccharification. The enzyme-producing strain harbored a synthetic genetic circuit to produce beta-glucosidase, which converts cellobiose to glucose, destroys itself via the lytic genes, and release the enzyme when the desired cell density is reached. The target chemical-producing strain was programmed by a synthetic genetic circuit to express enzymes in the synthetic metabolic pathway for isopropanol production when the enzyme-producing strain grows until release of the enzyme. Our results demonstrate the benefits of synthetic microbial consortia with distributed tasks for effective chemical production from biomass.

Improvement of 1,3-propanediol production using an engineered cyanobacterium, Synechococcus elongatus by optimization of the gene expression level of a synthetic metabolic pathway and production conditions.

The introduction of a synthetic metabolic pathway consisting of multiple genes derived from various organisms enables cyanobacteria to directly produce valuable chemicals from carbon dioxide. We previously constructed a synthetic metabolic pathway composed of genes from Escherichia coli, Saccharomyces cerevisiae, and Klebsiella pneumoniae. This pathway enabled 1,3-propanediol (1,3-PDO) production from cellular DHAP via glycerol in the cyanobacterium, Synechococcus elongatus PCC 7942. The production of 1,3-PDO (3.79mM, 0.29g/l) directly from carbon dioxide by engineered S. elongatus PCC 7942 was successfully accomplished. However, the constructed strain accumulated a remarkable amount of glycerol (12.6mM, 1.16g/l), an intermediate metabolite in 1,3-PDO production. Notably, enhancement of latter reactions of synthetic metabolic pathway for conversion of glycerol to 1,3-PDO increases 1,3-PDO production. In this study, we aimed to increase the observed 1,3-PDO production titer. First, the weaker S. elongatus PCC 7942 promoter, PLlacO1, was replaced with a stronger promoter (Ptrc) to regulate genes involved in the conversion of glycerol to 1,3-PDO. Second, the induction timing for gene expression and medium composition were optimized. Promoter replacement resulted in higher 1,3-PDO production than glycerol accumulation, and the amount of products (1,3-PDO and glycerol) generated via the synthetic metabolic pathway increased with optimization of medium composition. Accordingly, we achieved the highest titer of 1,3-PDO (16.1mM, 1.22g/l) and this was higher than glycerol accumulation (9.46mM, 0.87g/l). The improved titer was over 4-fold higher than that of our previous study.

Reconstruction of a metabolic regulatory network in Escherichia coli for purposeful switching from cell growth mode to production mode in direct GABA fermentation from glucose.

γ-aminobutyric acid (GABA) is a drug and functional food additive and is used as a monomer for producing the biodegradable plastic, polyamide 4. Recently, direct GABA fermentation from glucose has been developed as an alternative to glutamate-based whole cell bioconversion. Although total productivity in fermentation is determined by the specific productivity and cell amount responsible for GABA production, the optimal metabolic state for GABA production conflicts with that for bacterial cell growth. Herein, we demonstrated metabolic state switching from the cell growth mode based on the metabolic pathways of the wild type strain to a GABA production mode based on a synthetic metabolic pathway in Escherichia coli through rewriting of the metabolic regulatory network and pathway engineering. The GABA production mode was achieved by multiple strategies such as conditional interruption of the TCA and glyoxylate cycles, engineering of GABA production pathway including a bypass for precursor metabolite supply, and upregulation of GABA transporter. As a result, we achieved 3-fold improvement in total GABA production titer and yield (4.8g/L, 49.2% (mol/mol glucose)) in batch fermentation compared to the case without metabolic state switching (1.6g/L, 16.4% (mol/mol glucose)). This study reports the highest GABA production performance among previous reports on GABA fermentation from glucose using engineered E. coli.

Alcohol-tolerant mutants of cyanobacterium Synechococcus elongatus PCC 7942 obtained by single-cell mutant screening system.

Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2 , low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10 g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10 g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30 g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72 h incubation under light with or without 10 g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. © 2017 Wiley Periodicals, Inc.

Metabolic engineering of Synechococcus elongatus PCC 7942 for improvement of 1,3-propanediol and glycerol production based on in silico simulation of metabolic flux distribution.

BACKGROUND: Production directly from carbon dioxide by engineered cyanobacteria is one of the promising technologies for sustainable future. Previously, we have successfully achieved 1,3-propanediol (1,3-PDO) production using Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. The strain into which the synthetic metabolic pathway was introduced produced 3.48 mM (0.265 g/L) 1,3-PDO and 14.3 mM (1.32 g/L) glycerol during 20 days of incubation. In this study, the productivities of 1,3-PDO were improved by gene disruption selected by screening with in silico simulation. METHODS: First, a stoichiometric metabolic model was applied to prediction of cellular metabolic flux distribution in a 1,3-PDO-producing strain of S. elongatus PCC 7942. A genome-scale model of S. elongatus PCC 7942 constructed by Knoop was modified by the addition of a synthetic metabolic pathway for 1,3-PDO production. Next, the metabolic flux distribution predicted by metabolic flux balance analysis (FBA) was used for in silico simulation of gene disruption. As a result of gene disruption simulation, NADPH dehydrogenase 1 (NDH-1) complexes were found by screening to be the most promising candidates for disruption to improve 1,3-PDO production. The effect of disruption of the gene encoding a subunit of the NDH-1 complex was evaluated in the 1,3-PDO-producing strain. RESULTS AND CONCLUSIONS: During 20 days of incubation, the ndhF1-null 1,3-PDO-producing strain showed the highest titers: 4.44 mM (0.338 g/L) 1,3-PDO and 30.3 mM (2.79 g/L) glycerol. In this study, we successfully improved 1,3-PDO productivity on the basis of in silico simulation of gene disruption.

Cyanobacterial production of 1,3-propanediol directly from carbon dioxide using a synthetic metabolic pathway.

Production of chemicals directly from carbon dioxide using light energy is an attractive option for a sustainable future. The 1,3-propanediol (1,3-PDO) production directly from carbon dioxide was achieved by engineered Synechococcus elongatus PCC 7942 with a synthetic metabolic pathway. Glycerol dehydratase catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde in a coenzyme B12-dependent manner worked in S. elongatus PCC 7942 without addition of vitamin B12, suggesting that the intrinsic pseudovitamin B12 served as a substitute of coenzyme B12. The highest titers of 1,3-PDO (3.79±0.23 mM; 288±17.7 mg/L) and glycerol (12.62±1.55 mM; 1.16±0.14 g/L), precursor of 1,3-PDO, were reached after 14 days of culture under optimized conditions in this study.

A single cell culture system using lectin-conjugated magnetite nanoparticles and magnetic force to screen mutant cyanobacteria.

Cyanobacteria can be utilized as a potential biocatalyst for the production of biofuels and biochemicals directly from CO2. Useful mutants of cyanobacteria, which can grow rapidly or are resistant to specific metabolic products, are essential to improve the productivity of biofuels. In this study, we developed a single cell culture system to effectively screen mutant cyanobacteria using magnetite nanoparticles and magnetic force. Lens culinaris Agglutinin (LCA) was selected as a lectin, which binds to the surface of Synechococcus elongatus PCC7942 cells and the LCA-conjugated magnetite cationic liposomes (MCLs) were developed for magnetic labeling of PCC7942 cells. The MCL-labeled PCC7942 cells were magnetically patterned at a single cell level by using 6,400 iron pillars of the pin-holder device. The device enabled 1,600 single cells to be arrayed in one square centimeter. We cultured the patterned cells in liquid medium and achieved higher colony-forming ratio (78.4%) than that obtained using conventional solid culture method (4.8%). Single cells with different properties could be distinguished in the single cell culture system depending on their growth. Furthermore, we could selectively pick up the target cells and subsequently perform efficient isolation culture. The ratio of successful isolation culture using the developed method was 13 times higher than that of the conventional methods. Thus, the developed system would serve as a powerful tool for screening mutant cyanobacteria.

Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production.

Chemicals production by engineered microorganisms often requires induction of target gene expression at an appropriate cell density to reduce conflict with cell growth. The lux system in Vibrio fischeri is a well-characterized model for cell density-dependent regulation of gene expression termed quorum sensing (QS). However, there are currently no reports for application of the lux system to microbial chemical production. Here, we constructed a synthetic lux system as a tunable cell density sensor-regulator using a synthetic lux promoter and a positive feedback loop in Escherichia coli. In this system, self-induction of a target gene expression is driven by QS-signal, and its threshold cell density can be changed depending on the concentration of a chemical inducer. We demonstrate auto-redirection of metabolic flux from central metabolic pathways toward a synthetic isopropanol pathway at a desired cell density resulting in a significant increase in isopropanol production.

Enhancement of 3-hydroxypropionic acid production from glycerol by using a metabolic toggle switch.

BACKGROUND: 3-hydroxypropionic acid (3-HP) is an important platform for the production of C3 chemicals, including acrylic acid, methyl acrylate, and acrylamide. Microbial production of 3-HP is mainly due to glycerol metabolism. In this study, in order to improve microbial 3-HP production, we applied a metabolic toggle switch for controlling the glycerol metabolism to redirect the excess metabolic flux of central metabolic pathway toward an exogenous 3-HP producing pathway in Escherichia coli. RESULTS: The metabolic toggle switch enables conditional repression of the expression of a target gene during the fermentation. We individually performed conditional repression of glpK, tpiA, and gapA, which are involved in glycerol metabolism. The conditional repression of glpK and tpiA was not effective for 3-HP production under our experimental conditions. However, gapA conditional repression contributed to improve 3-HP production (titer, 54.2 ± 1.5 mM; yield, 32.1 ± 1.3 %) compared with that for the wild type strain. Additional deletion of endogenous yqhD, which is responsible for the production of a major byproduct, 1,3-propandiol, further increased 3-HP production (titer, 67.3 ± 2.1 mM; yield, 51.5 ± 3.2 %). The titer and yield were 80 and 94 % higher than those of the wild type strain, respectively. The obtained 3-HP yield from glycerol is comparable with the highest yield ever reported for microbial 3-HP production using glycerol as a sole carbon source. The measurement of intracellular metabolites showed the metabolic toggle switch successfully controlled the metabolic flux. CONCLUSION: The conditional repression of gapA by using the metabolic toggle switch combined with deletion of endogeneous yqhD increased 3-HP production approximately twofold from glycerol. This result indicates the metabolic toggle switch can be applied in various bio-production using diverse substrates.

Metabolic flux redirection from a central metabolic pathway toward a synthetic pathway using a metabolic toggle switch.

Overexpression of genes in production pathways and permanent knockout of genes in competing pathways are often employed to improve production titer and yield in metabolic engineering. However, the deletion of a pathway responsible for growth and cell maintenance has not previously been employed, even if its competition with the production pathway is obvious. In order to optimize intracellular metabolism at each fermentation phase for bacterial growth and production, a methodology employing conditional knockout is required. We constructed a metabolic toggle switch in Escherichia coli as a novel conditional knockout approach and applied it to isopropanol production. The resulting redirection of excess carbon flux caused by interruption of the TCA cycle via switching gltA OFF improved isopropanol production titer and yield up to 3.7 and 3.1 times, respectively. This approach is a useful tool to redirect carbon flux responsible for bacterial growth and/or cell maintenance toward a synthetic production pathway.

Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels.

Global energy and environmental problems have stimulated increased efforts towards synthesizing biofuels from renewable resources. Compared to the traditional biofuel, ethanol, higher alcohols offer advantages as gasoline substitutes because of their higher energy density and lower hygroscopicity. In addition, branched-chain alcohols have higher octane numbers compared with their straight-chain counterparts. However, these alcohols cannot be synthesized economically using native organisms. Here we present a metabolic engineering approach using Escherichia coli to produce higher alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol from glucose, a renewable carbon source. This strategy uses the host's highly active amino acid biosynthetic pathway and diverts its 2-keto acid intermediates for alcohol synthesis. In particular, we have achieved high-yield, high-specificity production of isobutanol from glucose. The strategy enables the exploration of biofuels beyond those naturally accumulated to high quantities in microbial fermentation.

Engineering a synthetic pathway in cyanobacteria for isopropanol production directly from carbon dioxide and light.

Production of alternate fuels or chemicals directly from solar energy and carbon dioxide using engineered cyanobacteria is an attractive method to reduce petroleum dependency and minimize carbon emissions. Here, we constructed a synthetic pathway composed of acetyl-CoA acetyl transferase (encoded by thl), acetoacetyl-CoA transferase (encoded by atoAD), acetoacetate decarboxylase (encoded by adc) and secondary alcohol dehydrogenase (encoded by adh) in Synechococcus elongatus strain PCC 7942 to produce isopropanol. The enzyme-coding genes, heterogeneously originating from Clostridium acetobutylicum ATCC 824 (thl and adc), Escherichia coli K-12 MG1655 (atoAD) and Clostridium beijerinckii (adh), were integrated into the S. elongatus genome. Under the optimized production conditions, the engineered cyanobacteria produced 26.5 mg/L of isopropanol after 9 days.

A hybrid in silico/in-cell controller for microbial bioprocesses with process-model mismatch.

Bioprocess optimization using mathematical models is prevalent, yet the discrepancy between model predictions and actual processes, known as process-model mismatch (PMM), remains a significant challenge. This study proposes a novel hybrid control system called the hybrid in silico/in-cell controller (HISICC) to address PMM by combining model-based optimization (in silico feedforward controller) with feedback controllers utilizing synthetic genetic circuits integrated into cells (in-cell feedback controller). We demonstrated the efficacy of HISICC using two engineered Escherichia coli strains, TA1415 and TA2445, previously developed for isopropanol (IPA) production. TA1415 contains a metabolic toggle switch (MTS) to manage the competition between cell growth and IPA production for intracellular acetyl-CoA by responding to external input of isopropyl ß-D-1-thiogalactopyranoside (IPTG). TA2445, in addition to the MTS, has a genetic circuit that detects cell density to autonomously activate MTS. The combination of TA2445 with an in silico controller exemplifies HISICC implementation. We constructed mathematical models to optimize IPTG input values for both strains based on the two-compartment model and validated these models using experimental data of the IPA production process. Using these models, we evaluated the robustness of HISICC against PMM by comparing IPA yields with two strains in simulations assuming various magnitudes of PMM in cell growth rates. The results indicate that the in-cell feedback controller in TA2445 effectively compensates for PMM by modifying MTS activation timing. In conclusion, the HISICC system presents a promising solution to the PMM problem in bioprocess engineering, paving the way for more efficient and reliable optimization of microbial bioprocesses.

Trace impurities in sodium phosphate influences the physiological activity of Escherichia coli in M9 minimal medium.

In the field of applied microbiology, reproducibility and experimental variability are important factors that influence both basic research as well as process development for industrial applications. Experimental reproducibility and accuracy depend not only on culture conditions such as temperature and aeration but also on raw materials and procedures used for media preparation. The M9 minimal medium is one of the most common synthetic media for culturing Escherichia coli and other bacteria. This synthetic medium can be used to observe and evaluate the physiological activity of microbes under minimal nutritional requirements and determine the limiting factor for the desired phenotype. Although one of the advantages using the M9 medium is that its composition can be modulated, it is difficult to control presence of trace components and impurities from the reagents for preparing this medium. Herein, we showed that trace ingredients present in the reagents used for M9 media preparation affect the bacterial physiological activities (e.g., cell growth, substrate consumption, and byproduct formation). Additionally, we systematically identified the trace ingredient that influenced phenotypic differences. Our results showed that the selection of reagents and accuracy during reagent preparation is important for experimental reproducibility in the field of bio-engineering and systems biology focused on the systematic and continuous development of biomolecular systems (e.g., biorefinery, metabolic engineering, and synthetic biology).

Dynamic metabolic engineering of Escherichia coli improves fermentation for the production of pyruvate and its derivatives.

Pyruvate is a key intermediate that is involved in various synthetic metabolic pathways for microbial chemical and fuel production. It is widely used in the food, chemical, and pharmaceutical industries. However, the microbial production of pyruvate and its derivatives compete with microbial cell growth, as pyruvate is an important metabolic intermediate that serves as a hub for various endogenous metabolic pathways, including gluconeogenesis, amino acid synthesis, TCA cycle, and fatty acid biosynthesis. To achieve a more efficient bioprocess for the production of pyruvate and its derivatives, it is necessary to reduce the metabolic imbalance between cell growth and target chemical production. For this purpose, we devised a dynamic metabolic engineering strategy within an Escherichia coli model, in which a metabolic toggle switch (MTS) was employed to redirect metabolic flux from the endogenous pathway toward the target synthetic pathway. Through a combination of TCA cycle interruption through MTS and reduction of pyruvate consumption in endogenous pathways, we achieved a drastic improvement (163 mM, 26-fold) in pyruvate production. In addition, we demonstrated the redirection of metabolic flux from excess pyruvate toward isobutanol production. The final isobutanol production titer of the strain harboring MTS was 26% improved compared with that of the control strain.

Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS).

The production of chemicals and fuels from renewable resources using engineered microbes is an attractive alternative for current fossil-dependent industries. Metabolic engineering has contributed to pathway engineering for the production of chemicals and fuels by various microorganisms. Recently, dynamic metabolic engineering harnessing synthetic biological tools has become a next-generation strategy in this field. The dynamic regulation of metabolic flux during fermentation optimizes metabolic states according to each fermentation stage such as cell growth phase and compound production phase. However, it is necessary to repeat the evaluation and redesign of the dynamic regulation system to achieve the practical use of engineered microbes. In this study, we performed quantitative metabolome analysis to investigate the effects of dynamic metabolic flux regulation on engineered Escherichia coli for γ-amino butyrate (GABA) fermentation. We prepared a stable isotope-labeled internal standard mixture (SILIS) for the stable isotope dilution method (SIDM), a mass spectrometry-based quantitative metabolome analysis method. We found multiple candidate bottlenecks for GABA production. Some metabolic reactions in the GABA production pathway should be engineered for further improvement in the direct GABA fermentation with dynamic metabolic engineering strategy.

Design of Synthetic Quorum Sensing Achieving Induction Timing-Independent Signal Stabilization for Dynamic Metabolic Engineering of E. coli.

Dynamic metabolic engineering that harnesses synthetic biological tools is a next-generation strategy for microbial chemical and fuel production. We previously reported a synthetic quorum sensing system combined with a metabolic toggle switch (QS-MTS) in E. coli. It autonomously redirected endogenous metabolic flux toward the synthetic metabolic pathway and improved biofuel production. However, its functions and effects on host metabolism were attenuated by induction timing delay. Here, we redesigned the QS-MTS to stabilize QS signaling efficiency and metabolic regulation. We performed a metabolome analysis to clarify the effects of QS-MTS redesign on host metabolism. We compared the contributions of conventional and redesigned QS-MTS to fed-batch fermentation. The redesigned QS-MTS was more conducive than the conventional QS-MTS to long-term processes such as fed-batch fermentation. Here, we present a circuit redesign for metabolic flux control based on dynamic characteristic evaluation and metabolome analysis.

An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study.

Significance of single cell measurements stems from the substantial temporal fluctuations and cell-cell variability possessed by individual cells. A major difficulty in monitoring surface non-adherent cells such as bacteria and yeast is that these cells tend to aggregate into clumps during growth, obstructing the tracking or identification of single-cells over long time periods. Here, we developed a microfluidic platform for long term single-cell tracking and cultivation with continuous media refreshing and dynamic chemical perturbation capability. The design highlights a simple device-assembly process between PDMS microchannel and agar membrane through conformal contact, and can be easily adapted by microbiologists for their routine laboratory use. The device confines cell growth in monolayer between an agar membrane and a glass surface. Efficient nutrient diffusion through the membrane and reliable temperature maintenance provide optimal growth condition for the cells, which exhibited fast exponential growth and constant distribution of cell sizes. More than 24 h of single-cell tracking was demonstrated on a transcription-metabolism integrated synthetic biological model, the gene-metabolic oscillator. Single cell morphology study under alcohol toxicity allowed us to discover and characterize cell filamentation exhibited by different E. coli isobutanol tolerant strains. We believe this novel device will bring new capabilities to quantitative microbiology, providing a versatile platform for single cell dynamic studies.