Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria

Baker, Joshua D.; Kysela, David T.; Zhou, Jinsheng; Madren, Seth; Wilkens, Andrew S.; Brun, Yves V.; Jacobson, Stephen C.

doi:10.1021/acs.analchem.6b00889

Cited by 16 publications

(15 citation statements)

References 35 publications

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“…The microfluidic device was fabricated through a combination of electron-beam lithography, contact photolithography, and polymer casting (52). Briefly, the microfluidic device is comprised of fluid and control layers both cast in poly(dimethylsiloxane) (PDMS) and a glass coverslip.…”

Section: Methodsmentioning

confidence: 99%

“…A period of adaptation following exposure to illumination was observed; thus, data analysis was restricted to periods of steady state. Cell identification and tracking were analyzed by a series of MATLAB programs (The MathWorks, Inc.) (52). The program extracted fluorescence intensity along a line profile down the longitudinal center of each sub-micron channel.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…Here, we use fluorescence microscopy and microfluidics to quantitatively measure parameters of B. subtilis FtsZ dynamics and cell division under the condition of chemostatic growth for extended periods of time (47–52). The automated poly(dimethylsiloxane) microfluidic system comprises a pneumatically actuated channel array of 600 channels having widths from 1.0 to 1.6 μm and heights of 1.0 μm to actively trap bacteria cells (52). Integrated pumps and valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading and nutrient flow, and the channel array confines bacterial growth to a single dimension, facilitating high-resolution, time-lapse imaging and tracking of individual cells over multiple generations.…”

Section: Introductionmentioning

confidence: 99%

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The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling inBacillus subtilis

Zhou

Dempwollf

et al. 2019

Preprint

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lack DNA and arise when cell division occurs, not at the midcell, but rather near one cell pole. 75Polar division was attributed to a mislocalization of FtsZ rings and the recruitment of the same 76 machinery that would ordinarily promote medial septation (20). The mutation responsible for 77 minicell formation was mapped to a genetic locus encoding the membrane-associated ATPase 78MinD and the 22). MinD is anchored to the membrane by an 79 amphipathic helix, and MinD recruits and activates MinC by direct interaction (23-30). MinC 80 binds to the C-terminus of FtsZ and destabilizes the FtsZ ring (31)(32)(33)(34)(35). In E. coli, the activity of 81 the MinCD complex is dynamically restricted to the polar region by oscillation in which MinCD 82 polar polymerization is antagonized by MinE-mediated depolymerization (36-40). In B. subtilis 83 however, the activity of the MinCD complex is statically restricted to membranes with high 84 curvature by MinJ/DivIVA, such that the entire 4-protein complex assembles at the invaginating 85 nascent division plane and remains at the cell poles after division (41)(42)(43)(44)(45)(46). 86Here, we use fluorescence microscopy and microfluidics to quantitatively measure 87 parameters of B. subtilis FtsZ dynamics and cell division under the condition of chemostatic 88 growth for extended periods of time (47-52). The automated poly(dimethylsiloxane) microfluidic 89 system comprises a pneumatically actuated channel array of 600 channels having widths from 90 1.0 to 1.6 m and heights of 1.0 m to actively trap bacteria cells (52). Integrated pumps and 91 valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading 92 and nutrient flow, and the channel array confines bacterial growth to a single dimension, 93 facilitating high-resolution, time-lapse imaging and tracking of individual cells over multiple 94 generations. In wild type cells, we find that Z-rings persist for a period greater than one cell 95 cycle because Z-rings transiently remain at the cell poles following septum completion. We 96 further show find that the primary function of the Min system is Z-ring disassembly such that in 97 the absence of Min, Z-rings persist longer than the duration of the experiment. Indefinite Z-ring 98 persistence results in cells with multiple Z-rings per compartment, and Z-ring must directly 99 compete for newly synthesized FtsZ monomers. Moreover, we show that min mutant cells are 100

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling inBacillus subtilis

Zhou

Dempwollf

et al. 2019

Preprint

Self Cite

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show abstract

“…Here, we use fluorescence microscopy and microfluidics to quantitatively measure parameters of B. subtilis FtsZ dynamics and cell division under the condition of chemostatic growth for extended periods of time (47)(48)(49)(50)(51)(52). The automated poly(dimethylsiloxane) microfluidic system comprises a pneumatically actuated array of 600 channels having widths of 1.0 to 1.6 m and heights of 1.0 m to actively trap bacterial cells (52). Integrated pumps and valves perform on-chip fluid and cell manipulations that provide dynamic control of cell loading and nutrient flow, and the channel array confines bacterial growth to a single dimension, facilitating high-resolution, time-lapse imaging and tracking of individual cells over multiple generations.…”

mentioning

confidence: 99%

The Min System Disassembles FtsZ Foci and Inhibits Polar Peptidoglycan Remodeling in Bacillus subtilis

Zhou

Dempwolff

et al. 2020

mBio

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A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis. Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling. IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the “inert” property of cell walls at the poles.

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High-throughput system-wide engineering and screening for microbial biotechnology

Vervoort

Linares

Roncoroni

et al. 2017

Current Opinion in Biotechnology

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Genetic engineering and screening of large number of cells or populations is a crucial bottleneck in today's systems biology and applied (micro)biology. Instead of using standard methods in bottles, flasks or 96-well plates, scientists are increasingly relying on high-throughput strategies that miniaturize their experiments to the nanoliter and picoliter scale and the single-cell level. In this review, we summarize different high-throughput system-wide genome engineering and screening strategies for microbes. More specifically, we will emphasize the use of multiplex automated genome evolution (MAGE) and CRISPR/Cas systems for high-throughput genome engineering and the application of (lab-on-chip) nanoreactors for high-throughput single-cell or population screening.

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Programmable, Pneumatically Actuated Microfluidic Device with an Integrated Nanochannel Array To Track Development of Individual Bacteria

Cited by 16 publications

References 35 publications

The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling inBacillus subtilis

The Min system disassembles FtsZ foci and inhibits polar peptidoglycan remodeling inBacillus subtilis

The Min System Disassembles FtsZ Foci and Inhibits Polar Peptidoglycan Remodeling in Bacillus subtilis

High-throughput system-wide engineering and screening for microbial biotechnology

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