单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

The overall goal of this procedure is to visualize and quantify the distinct subpopulations of specialized cells that differentiate in biofilms of the model organism, bacillus subtles. This is accomplished by first inserting transcriptional reporters into the chromosome of B subtles. These reporters will be expressed exclusively in the subpopulation of cells under study.

The next step is to promote the development of a biofilm by growing B subtles in the biofilm inducing medium, Ms GG gg. After biofilm formation, the cells from the biofilm are dispersed and fixed with para formaldehyde. The final step of the procedure is to detect the fluorescence signal emitted by single cells of the microbial community using fluorescence, microscopy and flow cytometry.

Ultimately, the results show how cells specialized to produce and secrete. The extracellular matrix that constitute the biofilm are a different subpopulation from the cells that secrete surfactant the signaling molecule responsible for the differentiation of the subpopulation of matrix producers. The main advantage of this technique over existing methods like quantification of transcription expression using Vita Ities assays of microray analysis, is that it allows us to monitor gene expression at a single cell level and visualize particularly small cell populations of cells there are specialized to express a specific gene.

These gene expressions will not be detected when analyzing the overall population. Integrative plasmids are used for labeling B subtles and an example is shown here. PTAA, the promoter of the genes responsible for the production of tass.

A matrix protein is cloned into the vector using the restriction sites ECO R one and Hindi three. The expression of the CFP reporter gene is now under the control of ptap A to begin the procedure for labeling B, subtles linearize the integrative plasmid by enzymatic digestion. The recommended restriction enzyme is XH oh one when the integrative plasmid is ready, induce natural competence in B subtlest strain 1 68 by growing the cells in competence inducing medium for five hours at 37 degrees Celsius.

Add the linearized plasmid into the culture of competent B subtlest cells and incubate for two hours at 37 degrees Celsius after two hours, plate the cells on agar with spect mycin and incubate overnight at 37 degrees Celsius. The B subtles chromosome has two neutral loci, Amy, E, E, and LAC A, which can be used to integrate reporter fusions without affecting the development of the biofilm. The linearized PKM 0 0 8 plasmid integrates into the genome of B subtles by double recombination.

The next step is to transfer the reporter from the labeled strain to a strain that is able to form biofilms using the SPP one phage transduction protocol. For the purpose of this video, only the important steps in the protocol are shown. Grow the donor strain 1 68, harboring the reporter fusion in TY medium at 37 degrees Celsius until the culture reaches early stationary phase at 200 microliters of cells to 100 microliters of diluted phage stock in a glass tube.

And let's stand at 37 degrees Celsius for 15 minutes. Next, pipette hot TY soft agar into the tube and mix thoroughly by vortexing. Gently spread onto a fresh TY plate and incubate at 37 degrees Celsius for eight to 16 hours to allow phage halos to arise.

To collect the phage halos, add three milliliters of TY medium to the plate and use the glass pipette to break up the top agar. Transfer the agar suspension to a 15 milliliter falcon tube. Vortex gently to break up the agar and then centrifuge at 1000 G for 15 minutes.

Transfer the supernatant to a new tube. Pass the supernatant through a point 22 micron syringe filter. Use this supernatant to infect a culture of the recipient strain grown in TY medium.

Add 30 microliters of supernatant to 10 milliliters of culture diluted one to 10, and incubate for 30 minutes at 37 degrees Celsius. After that centrifuge at 5, 000 RPM for seven minutes, resus suspend the pellet in 200 microliters of TY medium and spread on a plate containing SPECT mycin plus 10 millimolar. Sodium citrate.

Incubate at 37 degrees Celsius for 12 to 36 hours. Select a colony and grow it overnight in liquid LB at 37 degrees Celsius. Finally, spot three microliters of the overnight culture on solid biofilm inducing medium.

Allow cells to grow for 72 hours at 30 degrees Celsius. After three days of growth, biofilms formed on the surface of the Ms.GG agar will develop a complex morphological architecture in the surface of the agar. To begin this procedure, use a toothpick or tweezers to remove the biofilm from the surface of the MSGG agar.

The consistency of the biofilm should enable it to be peeled off from the surface of the agar. In one piece, place the biofilm in three milliliters of PBS buffer, disperse the biofilm by mild. Perform 12 pulses with an output of three and an amplitude of 0.7 seconds to fix the samples prior to single cell analysis.

Resus suspend 300 microliters of the cell suspension in one milliliter, a 4%paraform aldehyde solution, and incubate at room temperature for exactly seven minutes. After the seven minute fixation, wash cells in PBS buffer three times. Finally, resuspend cells in 300 microliters of PBS buffer prior to fluorescence microscopy.

Prepare a microscope slide by pipetting 200 microliters of 0.8%agros over the slide, and carefully covering it with another slide. After two minutes, remove the upper slide gently to obtain a layer of agros attached to the slide on the bottom spot. Two microliters of fixed cells on the surface of the agros layer and cover it with a microscope cover glass.

Place the sample on the stage of the fluorescence microscope. Set the excitation period according to a negative control that shows no fluorescence in the conditions selected for the experiment. The exposure should be between 50 to 200 milliseconds.

Acquire a fluorescence image as well as a bright field image for each field of view. To prepare cells for flow cytometry analysis, disperse the sample of fixed cells by mild sonication. Perform two series of 12 pulses with an output of five and an amplitude of 0.7 seconds to disperse clumps into single cells without causing cell lysis.

Confirm the efficiency of cell dispersion by light microscopy. Next, dilute the sample one to 100 in PBS buffer. A BD fax canto two flow cytometer will be used for this analysis for YFP fluorescence.

A laser excitation at 488 nanometers coupled with a 5 30 30 filter is used for CFP fluorescence. A laser excitation at 405 nanometers coupled with a 4 0 8 40 filter is used, calibrate the flow cytometer with two negative controls. A sample of PBS buffer with no cells in suspension should serve as a negative control for the size of the particle sensed by the flow cytometer.

A sample without a fluorescent reporter should serve as a negative control for the fluorescent sensitivity of the flow. Cytometer after calibration, place the sample labeled with a fluorescent reporter in the flow cytometer for each sample, analyze at least 50, 000 events with a flow rate between 303, 000 events per second. When B Subtles grows on a plate of the biofilm inducing medium, MSGG biofilm formation is observed after three days of incubation at 30 degrees Celsius.

The biofilm shows a complex morphological architecture that is indicative of the distinct participating cell subpopulations. For instance, production of the extracellular matrix and biofilms results in the formation of wrinkles on the surface of the colony. This figure shows an example of visualization of cell differentiation in a single labeled strain using fluorescence microscopy.

This strain harbors the fluorescent reporter Ptap, A CFP that is expressed in the subpopulation of matrix producing cells colored in blue. This subpopulation produces and secretes the extracellular matrix that constitutes the biofilm. This next image is the result of visualization by fluorescence microscopy of a double labeled strain that harbors the reporter Ptap, A CFP, and the additional reporter P Surfactin YFP.

This second reporter enables monitoring of the subpopulation of cells responsible for secreting the signaling molecule surfactin, which triggers the signaling cascade for the differentiation of the matrix producers. Here, the matrix producers are false colored in blue, while the surfactant producers are false colored in yellow, the results of 2D flow cytometry using a single labeled strain harboring the reporter ptap. A YFB is presented here.

The untreated control strain without any fluorescent protein genes showed a single population with low relative fluorescence cells harboring ptap. A YFP under non biofilm inducing conditions did not differentiate the subpopulation of matrix producers, and the whole population also showed low relative fluorescence. In contrast, under biofilm inducing conditions, a subpopulation of cells with a high relative fluorescence was detected as a shoulder to the right of the low relative fluorescence.

Peak results obtained from 3D flow cytometry are shown here. Fluorescence signals of the channels monitored are presented in the X axis for YFP and Y axis For CFP, the upper left panel shows a strain with no fluorescent protein genes as a control for background fluorescence. In the right upper panel, a single labeled strain was used to detect the subpopulation of surfactin producers In the YFP fluorescence channel framed in yellow.

In the bottom left panel, a different single labeled strain was used to detect the subpopulation of matrix producers. In the CFP fluorescence channel framed in blue, the subpopulations of matrix producers and surfactant producers are monitored using the double labeled strain and the results are presented. In this bottom right panel.

Two subpopulations of cells expressing high levels of the fluorescent reporters are detected. Each population is framed showing that there is no overlap in the expression of the reporters between the two subpopulations of specialized cells. This final figure shows the quantification of the subpopulations of matrix producers and cannibals by 3D flow cytometry.

In the upper right panel, a single labeled strain was used to detect the subpopulation of cannibals. In the YFP fluorescence channel framed in yellow, this subpopulation of cannibals has been described to differentiate coordinately with the subpopulation of matrix producers. In the bottom left panel, the subpopulation of matrix producers is detected in another single labeled strain in the CFP fluorescence channel.

Framed in blue, the results from the double labeled strain in the bottom right panel showed a single subpopulation of fluorescent cells expressing both YFP of CFP framed in green. The simultaneous expression of the two reporters indicates that both cell differentiation pathways are coordinate activated in the same subpopulation. After watching this video, you should have a good understanding of how to visualize and quantify the distinct subpopulations of cells that constitute a microbial biofilm using celling analysis of the cell population such as fluorescent microscopy or flow cytometry.