Three-dimensional imaging for the quantification of spatial patterns in microbiota of the intestinal mucosa

Improving our understanding of host-microbe relationships in the gut requires the ability to both visualize and quantify the spatial organization of microbial communities in their native orientation with the host tissue. We developed a systematic procedure to quantify the three-dimensional (3D) spatial structure of the native mucosal microbiota in any part of the intestines with taxonomic and high spatial resolution. We performed a 3D biogeographical analysis of the microbiota of mouse cecal crypts at different stages of antibiotic exposure. By tracking eubacteria and four dominant bacterial taxa, we found that the colonization of crypts by native bacteria is a dynamic and spatially organized process. Ciprofloxacin treatment drastically reduced bacterial loads and eliminated Muribaculaceae (or all Bacteroidetes entirely) even 10 d after recovery when overall bacterial loads returned to preantibiotic levels. Our 3D quantitative imaging approach revealed that the bacterial colonization of crypts is organized in a spatial pattern that consists of clusters of adjacent colonized crypts that are surrounded by unoccupied crypts, and that this spatial pattern is resistant to the elimination of Muribaculaceae or of all Bacteroidetes by ciprofloxacin. Our approach also revealed that the composition of cecal crypt communities is diverse and that Lactobacilli were found closer to the lumen than Bacteroidetes, Ruminococcaceae, and Lachnospiraceae, regardless of antibiotic exposure. Finally, we found that crypts communities with similar taxonomic composition were physically closer to each other than communities that were taxonomically different.Peer reviewe

On the synchronization of synthetic genetic oscillators in single cells and colonies

Synthetic and systems biology propose the rational construction and alteration of the molecular networks that govern life processes, with the purpose of understanding how their architecture relates to the emerging dynamics and for the creation of novel functions. This approach is made possible by the tools of modern molecular biology and the quantitative insights of physical sciences. In this context, microfluidic technology has become the tool of choice for our investigations. Our work is greatly motivated by the prominent role that oscillations play in organizing the dynamics of all living things and by the observation of self-organization of bacterial colonies. In Chapter 2 the basic physics of fluid flow and mass transport in microfluidic devices are presented along with several examples of the application of this technology to quantitative biology. In Chapter 3 I discuss a study on the entrainment of biological clocks through the use of a bacterial synthetic oscillator. In Chapter 4 we discuss the construction and characterization of a genetic oscillator that produces group oscillations. Finally, in Chapter 5 we discuss the dynamic mechanical interactions that arise from the spatially constrained growth of bacteri

3D Imaging for the Quantification of Spatial Patterns in Microbiota of the Intestinal Mucosa

Please scroll down to see download links. Data and scripts for "3D Imaging for the Quantification of Spatial Patterns in Microbiota of the Intestinal Mucosa" submitted as a preprint to bioRxiv and accepted to PNAS (doi pending). Contents (see also DataList document in download): ****Confocal imaging of bacteria, mucus and murine epithelium in the proximal colon of a specific-pathogen free mouse (Fig. 3A-F) Raw multi-color confocal imaging of bacteria embedded in mucus over folds in the epithelium of the proximal colon. ****Multiplexed, spectral confocal imaging of bacterial communities in the cecal crypts of a specific-pathogen free mouse (Fig. 5C-Q) Raw spectral imaging Linear unmixing of spectral imaging Processing and analysis of unmixed spectral imaging in arivis Vision4D 3.0 ****Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Spectral and standard confocal imaging of bacterial communities in cecal crypts at different stages of antibiotic exposure: unexposed (2 mice), antibiotic (3 mice), and recovery (3 mice). (Figs. 6-7) Raw spectral imaging Linear unmixing of spectral imaging Processing and analysis of unmixed spectral imaging in arivis Vision4D 3.0 ****Mouse unexposed to ciprofloxacin #1 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse unexposed to ciprofloxacin #2 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse exposed to ciprofloxacin #1 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse exposed to ciprofloxacin #2 Standard confocal imaging of cecum Confocal imaging of cecum processed in arivis Vision4D 3.0 ****Mouse exposed to ciprofloxacin #3 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse that recovered from ciprofloxacin for 10 days #1 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse that recovered from ciprofloxacin for 10 days #2 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0 ****Mouse that recovered from ciprofloxacin for 10 days #3 Raw spectral confocal imaging of cecum Spectral confocal imaging of cecum after linear unmixing Confocal imaging of cecum after linear unmixing, and processed in arivis Vision4D 3.0Related Publication: 3D Imaging for the Quantification of Spatial Patterns in Microbiota of the Intestinal Mucosa https://doi.org/10.1101/2021.10.07.463215 engFiles available via S3 at https://renc.osn.xsede.org/ini210004tommorrell/0_D1.20077/Antibiotic_01/AC1_20X_01.czi 4.98 GB Download AC1_20X_01_Linear%20unmixing.czi 0 GB Download AC1_20X_02.czi 5.04 GB Download AC1_20X_02_Linear%20unmixing.czi 0 GB Download AC1_20X_03.czi 5.6 GB Download AC1_20X_03_Linear%20unmixing.czi 0 GB Download AC1_20X_04.czi 5.63 GB Download AC1_20X_04_Linear%20unmixing.czi 0 GB Download AC1_20X_05.czi 6.13 GB Download AC1_20X_05_Linear%20unmixing.czi 0 GB Download AC1_20X_06.czi 6.12 GB Download AC1_20X_06_Linear%20unmixing.czi 0 GB Download AC1_20X_07.czi 6.45 GB Download AC1_20X_07_Linear%20unmixing.czi 0 GB Download AC1_20X_08.czi 5.16 GB Download AC1_20X_08_Linear%20unmixing.czi 0 GB Download AC1_20X_09.czi 4.98 GB Download AC1_20X_09_Linear%20unmixing.czi 0 GB Download AC1_20X_10_Linear%20unmixing.czi 0 GB Download AC1_vision4D.zip 4.99 GB Download AC2_20X_01.czi 0.25 GB Download AC2_20X_02.czi 0.18 GB Download 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S7_PT3_03.czi 5.62 GB Download S7_PT3_03_Linear%20unmixing.czi 0 GB Download S7_PT3_03_vision4D.zip 0.73 GB Download S14_01.czi 0.6 GB Download S14_02.czi 0.74 GB Download S14_03.czi 0.55 GB Download S14_04.czi 0.82 GB Download RC1_20X_01.czi 6.45 GB Download RC1_20X_01_Linear%20unmixing.czi 0 GB Download RC1_20X_02.czi 5.27 GB Download RC1_20X_02_Linear%20unmixing.czi 0 GB Download RC1_20X_03.czi 6.01 GB Download RC1_20X_03_Linear%20unmixing.czi 0 GB Download RC1_20X_04.czi 6.7 GB Download RC1_20X_04_Linear%20unmixing.czi 0 GB Download RC1_20X_05.czi 5.84 GB Download RC1_20X_05_Linear%20unmixing.czi 0 GB Download RC1_20X_06.czi 8.68 GB Download RC1_20X_06_Linear%20unmixing.czi 0 GB Download RC1_20X_07.czi 6.93 GB Download RC1_20X_07_Linear%20unmixing.czi 0 GB Download RC1_20X_08.czi 5.87 GB Download RC1_20X_08_Linear%20unmixing.czi 0 GB Download RC1_20X_09.czi 5.7 GB Download RC1_20X_09_Linear%20unmixing.czi 0 GB Download RC1_20X_10.czi 4.93 GB Download 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RC3_20X_02_Linear%20unmixing.czi 0 GB Download RC3_20X_03.czi 4.19 GB Download RC3_20X_03_Linear%20unmixing.czi 0 GB Download RC3_20X_04.czi 4.35 GB Download RC3_20X_04_Linear%20unmixing.czi 0 GB Download RC3_20X_05.czi 4.3 GB Download RC3_20X_05_Linear%20unmixing.czi 0 GB Download RC3_20X_06.czi 7.62 GB Download RC3_20X_06_Linear%20unmixing.czi 0 GB Download RC3_20X_07.czi 5.15 GB Download RC3_20X_07_Linear%20unmixing.czi 0 GB Download RC3_20X_08.czi 4.68 GB Download RC3_20X_08_Linear%20unmixing.czi 0 GB Download RC3_20X_09.czi 5.35 GB Download RC3_20X_09_Linear%20unmixing.czi 0 GB Download RC3_20X_10.czi 5.59 GB Download RC3_20X_10_Linear%20unmixing.czi 0 GB Download RC3_20X_11.czi 4.49 GB Download RC3_20X_11_Linear%20unmixing.czi 0 GB Download RC3_20X_12.czi 5.21 GB Download RC3_20X_12_Linear%20unmixing.czi 0 GB Download RC3_20X_13.czi 7.82 GB Download RC3_20X_13_Linear%20unmixing.czi 0 GB Download RC3_20X_14.czi 4.54 GB Download RC3_20X_14_Linear%20unmixing.czi 0 GB Download RC3_20X_15.czi 5.4 GB Download RC3_20X_15_Linear%20unmixing.czi 0 GB Download RC3_20X_16.czi 6.57 GB Download RC3_20X_16_Linear%20unmixing.czi 0 GB Download RC3_vision4D.zip 14.12 GB Download CM1_20X_01.czi 6.1 GB Download CM1_20X_01_Linear%20unmixing.czi 0 GB Download CM1_20X_02.czi 4.63 GB Download CM1_20X_02_Linear%20unmixing.czi 0 GB Download CM1_20X_03_Linear%20unmixing.czi 0 GB Download CM1_20X_04_Linear%20unmixing.czi 0 GB Download CM1_20X_05.czi 6.74 GB Download CM1_20X_05_Linear%20unmixing.czi 0 GB Download CM1_20X_06.czi 5.55 GB Download CM1_20X_06_Linear%20unmixing.czi 0 GB Download CM1_20X_07.czi 6.45 GB Download CM1_20X_07_Linear%20unmixing.czi 0 GB Download CM1_20X_08.czi 5.35 GB Download CM1_20X_08_Linear%20unmixing.czi 0 GB Download CM1_20X_09.czi 5.76 GB Download CM1_20X_09_Linear%20unmixing.czi 0 GB Download CM1_20X_10.czi 5.19 GB Download CM1_20X_10_Linear%20unmixing.czi 0 GB Download CM1_20X_11.czi 5.65 GB Download CM1_20X_11_Linear%20unmixing.czi 0 GB Download 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GB Download CM2_20X_09_Linear%20unmixing.czi 0 GB Download CM2_20X_10.czi 5.19 GB Download CM2_20X_10_Linear%20unmixing.czi 0 GB Download CM2_20X_11.czi 4.63 GB Download CM2_20X_11_Linear%20unmixing.czi 0 GB Download CM2_20X_12.czi 3.54 GB Download CM2_20X_12_Linear%20unmixing.czi 0 GB Download CM2_20X_13.czi 4.8 GB Download CM2_20X_13_Linear%20unmixing.czi 0 GB Download CM2_20X_14.czi 3.82 GB Download CM2_20X_14_Linear%20unmixing.czi 0 GB Download CM2_20X_15.czi 4.22 GB Download CM2_20X_15_Linear%20unmixing.czi 0 GB Download CM2_20X_16.czi 5.3 GB Download CM2_20X_16_Linear%20unmixing.czi 0 GB Download CM2_20X_17.czi 5.05 GB Download CM2_20X_17_Linear%20unmixing.czi 0 GB Download CM2_20X_18.czi 4.85 GB Download CM2_20X_18_Linear%20unmixing.czi 0 GB Download CM2_vision4D.zip 13.78 GB Downloa

DNA Assembly in 3D Printed Fluidics

The process of connecting genetic parts—DNA assembly—is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from $0.61 to$5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.United States. Dept. of Defense. Assistant Secretary of Defense for Research & Engineering (Air Force Contract FA8721-05-C-0002